Small cell lung cancer associated antigens and uses therefor

ABSTRACT

Cancer associated antigens have been identified by autologous antibody screening of libraries of nucleic acids expressed in small cell lung cancer cells using antisera from cancer patients. The invention relates to nucleic acids and encoded polypeptides which are cancer associated antigens expressed in patients afflicted with small cell lung cancer. The invention provides, among other things, isolated nucleic acid molecules, expression vectors containing those molecules and host cells transfected with those molecules. The invention also provides isolated proteins and peptides, antibodies to those proteins and peptides and cytotoxic T lymphocytes which recognize the proteins and peptides. Fragments of the foregoing including functional fragments and variants also are provided. Kits containing the foregoing molecules additionally are provided. The molecules provided by the invention can be used in the diagnosis, monitoring, research, or treatment of conditions characterized by the expression of one or more cancer associated antigens.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/489,101, filed Jan. 21, 2000, now abandoned, the disclosure of whichis incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to nucleic acids and encoded polypeptides whichare cancer associated antigens expressed in patients afflicted with avariety of cancers. The invention also relates to agents which bind thenucleic acids or polypeptides. The nucleic acid molecules, polypeptidescoded for by such molecules and peptides derived therefrom, as well asrelated antibodies and cytolytic T lymphocytes, are useful, inter alia,in diagnostic and therapeutic contexts.

BACKGROUND OF THE INVENTION

The mechanism by which T cells recognize foreign materials has beenimplicated in cancer. A number of cytolytic T lymphocyte (CTL) clonesdirected against autologous melanoma antigens, testicular antigens, andmelanocyte differentiation antigens have been described. In manyinstances, the antigens recognized by these clones have beencharacterized.

The use of autologous CTLs for identifying tumor antigens requires thatthe target cells which express the antigens can be cultured in vitro andthat stable lines of autologous CTL clones which recognize theantigen-expressing cells can be isolated and propagated. While thisapproach has worked well for melanoma antigens, other tumor types, suchas epithelial cancers including breast and colon cancer, have provedrefractory to the approach.

More recently another approach to the problem has been described bySahin et al. (Proc. Natl. Acad. Sci. USA 92:11810-11813, 1995).According to this approach, autologous antisera are used to identifyimmunogenic protein antigens expressed in cancer cells by screeningexpression libraries constructed from tumor cell cDNA. Antigen-encodingclones so identified have been found to have elicited an high-titerhumoral immune response in the patients from which the antisera wereobtained. Such a high-titer IgG response implies helper T cellrecognition of the detected antigen. These tumor antigens can then bescreened for the presence of MHC/HLA class I and class II motifs andreactivity with CTLs.

Presently there is a need for additional cancer antigens for developmentof therapeutics and diagnosis applicable to a greater number of cancerpatients having various cancers.

SUMMARY OF THE INVENTION

Autologous antibody screening has now been applied to small cell lungcancer using antisera from cancer patients. Numerous cancer associatedantigens have been identified. The invention provides, inter alia,isolated nucleic acid molecules, expression vectors containing thosemolecules and host cells transfected with those molecules. The inventionalso provides isolated proteins and peptides, antibodies to thoseproteins and peptides and CTLs which recognize the proteins andpeptides. Fragments including functional fragments and variants of theforegoing also are provided. Kits containing the foregoing moleculesadditionally are provided. The foregoing can be used in the diagnosis,monitoring, research, or treatment of conditions characterized by theexpression of one or more cancer associated antigens.

Prior to the present invention, only a handful of small cell lung cancerassociated genes had been identified in the past 20 years. The inventioninvolves the surprising discovery of several genes, some previouslyknown and some previously unknown, which are expressed in individualswho have cancer. These individuals all have serum antibodies against theproteins (or fragments thereof) encoded by these genes. Thus, abnormallyexpressed genes are recognized by the host's immune system and thereforecan form a basis for diagnosis, monitoring and therapy.

The invention involves the use of a single material, a plurality ofdifferent materials and even large panels and combinations of materials.For example, a single gene, a single protein encoded by a gene, a singlefunctional fragment thereof, a single antibody thereto, etc. can be usedin methods and products of the invention. Likewise, pairs, groups andeven panels of these materials and optionally other cancer associatedantigen genes and/or gene products can be used for diagnosis, monitoringand therapy. The pairs, groups or panels can involve 2, 3, 4, 5 or moregenes, gene products, fragments thereof or agents that recognize suchmaterials. A plurality of such materials are not only useful inmonitoring, typing, characterizing and diagnosing cells abnormallyexpressing such genes, but a plurality of such materials can be usedtherapeutically. An example of the use of a plurality of such materialsfor the prevention, delay of onset, amelioration, etc. of cancer cells,which express or will express such genes prophylactically or acutely.Any and all combinations of the genes, gene products, and materialswhich recognize the genes and gene products can be tested and identifiedfor use according to the invention. It would be far too lengthy torecite all such combinations; those skilled in the art, particularly inview of the teaching contained herein, will readily be able to determinewhich combinations are most appropriate for which circumstances.

As will be clear from the following discussion, the invention has invivo and in vitro uses, including for therapeutic, diagnostic,monitoring and research purposes. One aspect of the invention is theability to fingerprint a cell expressing a number of the genesidentified according to the invention by, for example, quantifying theexpression of such gene products. Such fingerprints will becharacteristic, for example, of the stage of the cancer, the type of thecancer, or even the effect in animal models of a therapy on a cancer.Cells also can be screened to determine whether such cells abnormallyexpress the genes identified according to the invention.

The invention, in one aspect, is a method of diagnosing a disordercharacterized by expression of a cancer associated antigen precursorcoded for by a nucleic acid molecule. The method involves the steps ofcontacting a biological sample isolated from a subject with an agentthat specifically binds to the nucleic acid molecule, an expressionproduct thereof, or a fragment of an expression product thereofcomplexed with an MHC, preferably an HLA, molecule, wherein the nucleicacid molecule is a NA Group 1 nucleic acid molecule, and determining theinteraction between the agent and the nucleic acid molecule, theexpression product or fragment of the expression product as adetermination of the disorder.

In one embodiment the agent is selected from the group consisting of (a)a nucleic acid molecule comprising NA Group 1 nucleic acid molecules ora fragment thereof, (b) a nucleic acid molecule comprising NA Group 3nucleic acid molecules or a fragment thereof, (c) a nucleic acidmolecule comprising NA Group 5 nucleic acid molecules or a fragmentthereof, (d) an antibody that binds to an expression product, or afragment thereof, of NA group 1 nucleic acids, (e) an antibody thatbinds to an expression product, or a fragment thereof, of NA group 3nucleic acids, (f) an antibody that binds to an expression product, or afragment thereof, of NA group 5 nucleic acids, (g) and agent that bindsto a complex of an MHC, preferably HLA, molecule and a fragment of anexpression product of a NA Group 1 nucleic acid, (h) an agent that bindsto a complex of an MHC, preferably HLA, molecule and a fragment of anexpression product of a NA group 3 nucleic acid, and (i) an agent thatbinds to a complex of an MHC, preferably HLA, molecule and a fragment ofan expression product of a NA Group 5 nucleic acid.

The disorder may be characterized by expression of a plurality of cancerassociated antigen precursors. Thus the methods of diagnosis may includeuse of a plurality of agents, each of which is specific for a differenthuman cancer associated antigen precursor (including at least one of thecancer associated antigen precursors disclosed herein), and wherein saidplurality of agents is at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9 or at least 10 suchagents.

In each of the above embodiments the disorder preferably is selectedfrom the group consisting of lung cancers including small cell lungcancer and non-small cell lung cancer, melanoma, colon cancer, breastcancer, head and neck cancer, transitional cancer, leiomyosarcoma andsynovial sarcoma.

In some embodiments, the nucleic acid molecule is selected from thegroup consisting of SOX2 nucleic acids, SOXI nucleic acids, ZIC2 nucleicacids, SOX3 nucleic acids and SOX21 nucleic acids. Preferably thenucleic acid molecule is selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11 and SEQ ID NO:12.

In certain embodiments, the biological sample is isolated from a tissueselected from the group consisting of non-brain, non-testis,non-prostate, non-small intestine and non-colon tissues.

In another aspect the invention is a method for determining regression,progression or onset of a condition characterized by expression ofabnormal levels of a protein encoded by a nucleic acid molecule that isa NA Group 1 molecule. The method involves the steps of monitoring asample, from a subject who has or is suspected of having the condition,for a parameter selected from the group consisting of (i) the protein,(ii) a peptide derived from the protein, (iii) an antibody whichselectively binds the protein or peptide, and (iv) cytolytic T cellsspecific for a complex of the peptide derived from the protein and anMHC molecule, as a determination of regression, progression or onset ofsaid condition. In one embodiment the sample is a body fluid, a bodyeffusion or a tissue.

In another embodiment the step of monitoring comprises contacting thesample with a detectable agent selected from the group consisting of (a)an antibody which selectively binds the protein of (i), or the peptideof (ii), (b) a protein or peptide which binds the antibody of (iii), and(c) a cell which presents the complex of the peptide and MHC molecule of(iv). In a preferred embodiment the antibody, the protein, the peptideor the cell is labeled with a radioactive label or an enzyme. The samplein a preferred embodiment is assayed for the peptide.

According to another embodiment the nucleic acid molecule is one of thefollowing: a NA Group 3 molecule or a NA Group 5 molecule. In stillanother embodiment, the nucleic acid molecule is selected from the groupconsisting of SOX2 nucleic acids, SOX1 nucleic acids, ZIC2 nucleicacids, SOX3 nucleic acids and SOX21 nucleic acids. Preferably thenucleic acid molecule is selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11 and SEQ ID NO:12.

In yet another embodiment the protein is a plurality of proteins, theparameter is a plurality of parameters, each of the plurality ofparameters being specific for a different of the plurality of proteins,at least one of which is a cancer associated protein encoded by a NAgroup 1 molecule. In certain embodiments the protein is a plurality ofproteins, at least one of which is encoded by SOX2 (SEQ ID NO:3) or ZIC2(SEQ ID NO:5), and wherein the parameter is a plurality of parameters,each of the plurality of parameters being specific for a different ofthe plurality of proteins.

The invention in another aspect is a pharmaceutical preparation for ahuman subject. The pharmaceutical preparation includes an agent whichwhen administered to the subject enriches selectively the presence ofcomplexes of an HLA molecule and a human cancer associated antigen, anda pharmaceutically acceptable carrier, wherein the human cancerassociated antigen is a fragment of a human cancer associated antigenprecursor encoded by a nucleic acid molecule which comprises a NA Group1 molecule. In one embodiment the nucleic acid molecule is a NA Group 3nucleic acid molecule or a NA group 5 nucleic acid molecule.

The agent in one embodiment comprises a plurality of agents, each ofwhich enriches selectively in the subject complexes of an HLA moleculeand a different human cancer associated antigen. Preferably theplurality is at least two, at least three, at least four or at least 5different such agents.

In certain embodiments, the agent comprises a plurality of agents, atleast one of which is a nucleic acid molecule selected from the groupconsisting of SOX2 nucleic acids, SOX1 nucleic acids, ZIC2 nucleicacids, SOX3 nucleic acids and SOX21 nucleic acids, and preferably atleast one of which is a nucleic acid molecule selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11 andSEQ ID NO:12, or an expression product thereof, and each of whichenriches selectively in the subject complexes of an HLA molecule and adifferent human cancer associated antigen.

In another embodiment the agent is selected from the group consisting of(1) an isolated polypeptide comprising the human cancer associatedantigen, or a functional variant thereof, (2) an isolated nucleic acidoperably linked to a promoter for expressing the isolated polypeptide,or functional variant thereof, (3) a host cell expressing the isolatedpolypeptide, or functional variant thereof, and (4) isolated complexesof the polypeptide, or functional variants thereof, and an HLA molecule.

The agent may be a cell expressing an isolated polypeptide. In oneembodiment the agent is a cell expressing an isolated polypeptidecomprising the human cancer associated antigen or a functional variantthereof. In another embodiment the agent is a cell expressing anisolated polypeptide comprising the human cancer associated antigen or afunctional variant thereof, and wherein the cell expresses an HLAmolecule that binds the polypeptide. The cell can express one or both ofthe polypeptide and HLA molecule recombinantly. In preferred embodimentsthe cell is nonproliferative. In other preferred embodiments, theisolated polypeptide is or includes a polypeptide encoded by a nucleicacid molecule selected from the group consisting of SOX2 nucleic acids,SOX1 nucleic acids, ZIC2 nucleic acids, SOX3 nucleic acids and SOX21nucleic acids, and preferably at least one of which is a nucleic acidmolecule selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:11 and SEQ ID NO:12. In yet another embodimentthe agent is at least two, at least three, at least four or at leastfive different polypeptides, each representing a different human cancerassociated antigen or functional variant thereof.

The agent in one embodiment is a PP Group 2 polypeptide. In otherembodiments the agent is a PP Group 3 polypeptide or a PP Group 4polypeptide.

In an embodiment each of the pharmaceutical preparations describedherein also includes an adjuvant.

According to another aspect the invention, a composition is providedwhich includes an isolated agent that binds selectively a PP Group 1polypeptide. In separate embodiments the agent binds selectively to apolypeptide selected from the following: a PP Group 2 polypeptide, a PPGroup 3 polypeptide, a PP Group 4 polypeptide, and a PP Group 5polypeptide. In other embodiments, the agent is a plurality of differentagents that bind selectively at least two, at least three, at leastfour, or at least five different such polypeptides. In each of the abovedescribed embodiments the agent may be an antibody. In a preferredembodiment, at least one of polypeptides is encoded by a nucleic acidmolecule selected from the group consisting of SOX2 nucleic acids, SOX1nucleic acids, ZIC2 nucleic acids, SOX3 nucleic acids and SOX21 nucleicacids, and preferably at least one of which is a nucleic acid moleculeselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:11 and SEQ ID NO:12, or a fragment thereof.

In another aspect the invention is a composition of matter composed of aconjugate of the agent of the above-described compositions of theinvention and a therapeutic or diagnostic agent. Preferably theconjugate is of the agent and a therapeutic or diagnostic that is atoxin, particularly an antineoplastic.

The invention in another aspect is a pharmaceutical composition whichincludes an isolated nucleic acid molecule selected from the groupconsisting of: (1) NA Group 1 molecules, and (2) NA Group 2 molecules,and a pharmaceutically acceptable carrier. In one embodiment theisolated nucleic acid molecule comprises a NA Group 3 or NA Group 4molecule. In another embodiment the isolated nucleic acid moleculecomprises at least two isolated nucleic acid molecules coding for twodifferent polypeptides, each polypeptide comprising a different cancerassociated antigen. In preferred embodiments, at least one of thepolypeptides is encoded by a nucleic acid molecule selected from thegroup consisting of SOX2 nucleic acids, SOX1 nucleic acids, ZIC2 nucleicacids, SOX3 nucleic acids and SOX21 nucleic acids, and preferably atleast one of which is a nucleic acid molecule selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11 andSEQ ID NO:12.

Preferably the pharmaceutical composition also includes an expressionvector with a promoter operably linked to the isolated nucleic acidmolecule. In another embodiment the pharmaceutical composition alsoincludes a host cell recombinantly expressing the isolated nucleic acidmolecule.

According to another aspect of the invention a pharmaceuticalcomposition is provided. The pharmaceutical composition includes anisolated polypeptide comprising a PP Group 1 or a PP Group 2polypeptide, and a pharmaceutically acceptable carrier. In oneembodiment the isolated polypeptide comprises a PP Group 3 or a PP Group4 polypeptide.

In another embodiment the isolated polypeptide comprises at least twodifferent polypeptides, each comprising a different cancer associatedantigen at least one of which is encoded by a NA group 1 molecule asdisclosed herein. In certain embodiments at least one of thepolypeptides is encoded by a nucleic acid molecule selected from thegroup consisting of SOX2 nucleic acids, SOX1 nucleic acids, ZIC2 nucleicacids, SOX3 nucleic acids and SOX21 nucleic acids, and preferably atleast one of which is a nucleic acid molecule selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11 andSEQ ID NO:12. In separate embodiments the isolated polypeptides areselected from the following: PP Group 3 polypeptides or HLA bindingfragments thereof and PP Group 5 polypeptides or HLA binding fragmentsthereof.

In an embodiment each of the pharmaceutical compositions describedherein also includes an adjuvant.

Another aspect the invention is an isolated nucleic acid moleculecomprising a NA Group 3 molecule. Another aspect the invention is anisolated nucleic acid molecule comprising a NA Group 4 molecule.

The invention in another aspect is an isolated nucleic acid moleculeselected from the group consisting of (a) a fragment of a nucleic acidselected from the group of nucleic acid molecules consisting of SEQ IDNos numbered below and comprising all nucleic acid sequences among SEQID Nos:3-17, of sufficient length to represent a sequence unique withinthe human genome, and identifying a nucleic acid encoding a human cancerassociated antigen precursor, (b) complements of (a), provided that thefragment includes a sequence of contiguous nucleotides which is notidentical to any sequence selected from the sequence group consisting of(1) sequences having the GenBank accession numbers of Table 4, (2)complements of (1), and (3) fragments of (1) and (2).

In one embodiment the sequence of contiguous nucleotides is selectedfrom the group consisting of: (1) at least two contiguous nucleotidesnonidentical to the sequences in Table 4, (2) at least three contiguousnucleotides nonidentical to the sequences in Table 4, (3) at least fourcontiguous nucleotides nonidentical to the sequences in Table 4, (4) atleast five contiguous nucleotides nonidentical to the sequences in Table4, (5) at least six contiguous nucleotides nonidentical to the sequencesin Table 4, or (6) at least seven contiguous nucleotides nonidentical tothe sequences in Table 4.

In another embodiment the fragment has a size selected from the groupconsisting of at least: 8 nucleotides, 10 nucleotides, 12 nucleotides,14 nucleotides, 16 nucleotides, 18 nucleotides, 20, nucleotides, 22nucleotides, 24 nucleotides, 26 nucleotides, 28 nucleotides, 30nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 200nucleotides, 1000 nucleotides and every integer length therebetween.

In yet another embodiment the molecule encodes a polypeptide which, or afragment of which, binds a human HLA receptor or a human antibody.

Another aspect of the invention is an expression vector comprising anisolated nucleic acid molecule of the invention described above operablylinked to a promoter.

According to one aspect the invention is an expression vector comprisinga nucleic acid operably linked to a promoter, wherein the nucleic acidis a NA Group 1 or Group 2 molecule. In another aspect the invention isan expression vector comprising a NA Group 1 or Group 2 molecule and anucleic acid encoding an MHC, preferably HLA, molecule.

In yet another aspect the invention is a host cell transformed ortransfected with an expression vector of the invention described above.

In another aspect the invention is a host cell transformed ortransfected with an expression vector comprising an isolated nucleicacid molecule of the invention described above operably linked to apromoter, or an expression vector comprising a nucleic acid operablylinked to a promoter, wherein the nucleic acid is a NA Group 1 or 2molecule and further comprising a nucleic acid encoding HLA.

According to another aspect of the invention an isolated polypeptideencoded by the isolated nucleic acid molecules the invention, describedabove, is provided. These include PP Group 1-5 polypeptides. Theinvention also includes a fragment of the polypeptide which isimmunogenic. In one embodiment the fragment, or a portion of thefragment, binds HLA or a human antibody. In still another aspect theinvention provides as isolated polypeptide comprising a fragment of apolypeptide selected from the group consisting of ZIC2, SOX1, SOX2, SOX3and SOX21 polypeptides, which is immunogenic, wherein the polypeptide isnot a full-length ZIC1, SOX1, SOX2, SOX3 or SOX21 polypeptide.

The invention includes in another aspect an isolated fragment of a humancancer associated antigen precursor which, or portion of which, bindsHLA or a human antibody, wherein the precursor is encoded by a nucleicacid molecule that is a NA Group 1 molecule. In one embodiment thefragment is part of a complex with HLA. In another embodiment thefragment is between 8 and 12 amino acids in length. In anotherembodiment the invention includes an isolated polypeptide comprising afragment of the polypeptide of sufficient length to represent a sequenceunique within the human genome and identifying a polypeptide that is ahuman cancer associated antigen precursor.

According to another aspect of the invention a kit for detecting thepresence of the expression of a cancer associated antigen precursor isprovided. The kit includes a pair of isolated nucleic acid moleculeseach of which consists essentially of a molecule selected from the groupconsisting of (a) a 12-32 nucleotide contiguous segment of thenucleotide sequence of any of the NA Group 1 molecules and (b)complements of (“a”), wherein the contiguous segments arenonoverlapping. In one embodiment the pair of isolated nucleic acidmolecules is constructed and arranged to selectively amplify an isolatednucleic acid molecule that is a NA Group 3 molecule. Preferably, thepair amplifies a human NA Group 3 molecule.

According to another aspect of the invention a method for treating asubject with a disorder characterized by expression of a human cancerassociated antigen precursor is provided. The method includes the stepof administering to the subject an amount of an agent, which enrichesselectively in the subject the presence of complexes of an HLA moleculeand a human cancer associated antigen, effective to ameliorate thedisorder, wherein the human cancer associated antigen is a fragment of ahuman cancer associated antigen precursor encoded by a nucleic acidmolecule selected from the group consisting of (a) a nucleic acidmolecule comprising NA group 1 nucleic acid molecules, (b) a nucleicacid molecule comprising NA group 3 nucleic acid molecules, (c) anucleic acid molecule comprising NA group 5 nucleic acid molecules.

In one embodiment the disorder is characterized by expression of aplurality of human cancer associated antigen precursors and wherein theagent is a plurality of agents, each of which enriches selectively inthe subject the presence of complexes of an HLA molecule and a differenthuman cancer associated antigen. Preferably the plurality is at least 2,at least 3, at least 4, or at least 5 such agents. In a preferredembodiment, at least one of the human cancer ssociated antigens is apolypeptide encoded by a nucleic acid molecule selected from the roupconsisting of SOX2 nucleic acids, SOX1 nucleic acids, ZIC2 nucleicacids, SOX3 nucleic acids and SOX21 nucleic acids, and preferably atleast one of which is a nucleic acid molecule selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11 andSEQ ID NO:12, or a fragment thereof.

In another embodiment the agent is an isolated polypeptide selected fromthe group consisting of PP Group 1, PP Group 2, PP Group 3, PP Group 4,and PP group 5 polypeptides.

In yet another embodiment the disorder is cancer.

According to another aspect the invention is a method for treating asubject having a condition characterized by expression of a cancerassociated antigen precursor in cells of the subject. The methodincludes the steps of (i) removing an immunoreactive cell containingsample from the subject, (ii) contacting the immunoreactive cellcontaining sample to the host cell under conditions favoring productionof cytolytic T cells against a human cancer associated antigen which isa fragment of the precursor, (iii) introducing the cytolytic T cells tothe subject in an amount effective to lyse cells which express the humancancer associated antigen, wherein the host cell is transformed ortransfected with an expression vector comprising an isolated nucleicacid molecule operably linked to a promoter, the isolated nucleic acidmolecule being selected from the group of nucleic acid moleculesconsisting of NA Group 1, NA Group 2, NA Group 3, NA Group 4, NA Group5.

In one embodiment the host cell recombinantly expresses an HLA moleculewhich binds the human cancer associated antigen. In another embodimentthe host cell endogenously expresses an HLA molecule which binds thehuman cancer associated antigen.

The invention includes in another aspect a method for treating a subjecthaving a condition characterized by expression of a cancer associatedantigen precursor in cells of the subject. The method includes the stepsof (i) identifying a nucleic acid molecule expressed by the cellsassociated with said condition, wherein said nucleic acid molecule is aNA Group 1 molecule (ii) transfecting a host cell with a nucleic acidselected from the group consisting of (a) the nucleic acid moleculeidentified, (b) a fragment of the nucleic acid identified which includesa segment coding for a cancer associated antigen, (c) deletions,substitutions or additions to (a) or (b), and (d) degenerates of (a),(b), or (c); (iii) culturing said transfected host cells to express thetransfected nucleic acid molecule, and; (iv) introducing an amount ofsaid host cells or an extract thereof to the subject effective toincrease an immune response against the cells of the subject associatedwith the condition. Preferably, the antigen is a human antigen and thesubject is a human. In certain preferred embodiments the nucleic acidmolecule is selected from the group consisting of SOX2 nucleic acids,SOX1 nucleic acids, ZIC2 nucleic acids, SOX3 nucleic acids and SOX21nucleic acids, and preferably at least one of which is a nucleic acidmolecule is selected from the group consisting of SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:11 and SEQ ID NO:12.

In one embodiment the method also includes the step of (a) identifyingan MHC molecule which presents a portion of an expression product of thenucleic acid molecule, wherein the host cell expresses the same MHCmolecule as identified in (a) and wherein the host cell presents an MHCbinding portion of the expression product of the nucleic acid molecule.

In another embodiment the method also includes the step of treating thehost cells to render them non-proliferative.

In yet another embodiment the immune response comprises a B-cellresponse or a T cell response. Preferably the response is a T-cellresponse which comprises generation of cytolytic T-cells specific forthe host cells presenting the portion of the expression product of thenucleic acid molecule or cells of the subject expressing the humancancer associated antigen.

In another embodiment the nucleic acid molecule is a NA Group 3molecule.

Another aspect of the invention is a method for treating or diagnosingor monitoring a subject having a condition characterized by expressionof an abnormal amount of a protein encoded by a nucleic acid moleculethat is a NA Group 1 molecule. The method includes the step ofadministering to the subject an antibody which specifically binds to theprotein or a peptide derived therefrom, the antibody being coupled to atherapeutically useflil agent, in an amount effective to treat thecondition.

In one embodiment the antibody is a monoclonal antibody. Preferably themonoclonal antibody is a chimeric antibody or a humanized antibody.

In another aspect the invention is a method for treating a conditioncharacterized by expression in a subject of abnormal amounts of aprotein encoded by a nucleic acid molecule that is a NA Group 1 nucleicacid molecule. The method involves the step of administering to asubject at least one of the pharmaceutical compositions of the inventiondescribed above in an amount effective to prevent, delav the onset of,or inhibit the condition in the subject. In one embodiment the conditionis cancer. In another embodiment the method includes the step of firstidentifying that the subject expresses in a tissue abnormal amounts ofthe protein.

The invention in another aspect is a method for treating a subjecthaving a condition characterized by expression of abnormal amounts of aprotein encoded by a nucleic acid molecule that is a NA Group 1 nucleicacid molecule. The method includes the steps of (i) identifying cellsfrom the subject which express abnormal amounts of the protein; (ii)isolating a sample of the cells; (iii) cultivating the cells, and (iv)introducing the cells to the subject in an amount effective to provokean immune response against the cells.

In one embodiment the method includes the step of rendering the cellsnon-proliferative, prior to introducing them to the subject.

In another aspect the invention is a method for treating a pathologicalcell condition characterized by abnormal expression of a protein encodedby a nucleic acid molecule that is a NA Group 1 nucleic acid molecule.The method includes the step of administering to a subject in needthereof an effective amount of an agent which inhibits the expression oractivity of the protein.

In one embodiment the agent is an inhibiting antibody which selectivelybinds to the protein and wherein the antibody is a monoclonal antibody,a chimeric antibody, a humanized antibody or a fragment thereof. Inanother embodiment the agent is an antisense nucleic acid molecule whichselectively binds to the nucleic acid molecule which encodes theprotein. In yet another important embodiment the nucleic acid moleculeis a NA Group 3 nucleic acid molecule. In other preferred embodiments,the nucleic acid molecule is a nucleic acid molecule selected from thegroup consisting of SOX2 nucleic acids, SOX1 nucleic acids, ZIC2 nucleicacids, SOX3 nucleic acids and SOX21 nucleic acids, and preferably atleast one of which is a nucleic acid molecule selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11 andSEQ ID NO:12.

The invention includes in another aspect a composition of matter usefulin stimulating an immune response to a plurality of proteins encoded bynucleic acid molecules that are NA Group 1 molecules. The composition isa plurality of peptides derived from the amino acid sequences of theproteins, wherein the peptides bind to one or more MHC moleculespresented on the surface of the cells which express an abnormal amountof the protein. In preferred embodiments, at least one of the proteinsis encoded by a nucleic acid molecule selected from the group consistingof SOX2 nucleic acids, SOX1 nucleic acids, ZIC2 nucleic acids, SOX3nucleic acids and SOX21 nucleic acids, and preferably at least one ofwhich is a nucleic acid molecule selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11 and SEQ ID NO:12.

In one embodiment at least a portion of the plurality of peptides bindto MHC molecules and elicit a cytolytic response thereto. In anotherembodiment the composition of matter includes an adjuvant. In anotherembodiment the adjuvant is a saponin, GM-CSF, or an interleukin. Instill another embodiment, the compositions also includes at least onepeptide useful in stimulating an immune response to at least one proteinwhich is not encoded by nucleic acid molecules that are NA Group 1molecules, wherein the at least one peptide binds to one or more MHCmolecules.

According to another aspect the invention is an isolated antibody whichselectively binds to a complex of: (i) a peptide derived from a proteinencoded by a nucleic acid molecule that is a NA Group 1 molecule and(ii) and an MHC molecule to which binds the peptide to form the complex,wherein the isolated antibody does not bind to (i) or (ii) alone.

In one embodiment the antibody is a monoclonal antibody, a chimericantibody, a humanized antibody or a fragment thereof.

The invention also involves the use of the genes, gene products,fragments thereof, agents which bind thereto, and so on in thepreparation of medicaments. A particular medicament is for treatingcancer and a more particular medicament is for treating small cell lungcancer.

For all of the foregoing, preferred disorders include cancers,particularly lung cancers including small cell lung cancer and non-smallcell lung cancer, melanoma, colon cancer, breast cancer, head and neckcancer, transitional cancer, leiomyosarcoma and synovial sarcoma.Preferred tissues include non-brain, non-testis, non-prostate, non-smallintestine and non-colon tissues.

These and other aspects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows the alignment of predicted protein sequences of SOX1, 2, 3and 21 (GenBank accession numbers O00570, P48431, P41225, AAC95381.1,respectively; SEQ ID Nos:18-21). Sequences encoded within theSEREX-isolated clones are in bold face type, and sequences absent inthese clones are in gray italics. The DNA-binding HMG domain is boxed.Amino acids identical between three and four SOX proteins arehighlighted in two shades of gray.

DETAILED DESCRIPTION OF THE INVENTION

In the above summary and in the ensuing description, lists of sequencesare provided. The lists are meant to embrace each single sequenceseparately, two or more sequences together where they form a part of thesame gene, any combination of two or more sequences which relate todifferent genes, including and up to the total number on the list, as ifeach and every combination were separately and specifically enumerated.Likewise, when mentioning fragment size, it is intended that a rangeembrace the smallest fragment mentioned to the full-length of thesequence (less one nucleotide or amino acid so that it is a fragment),each and every fragment length intended as if specifically enumerated.Thus, if a fragment could be between 10 and 15 in length, it isexplicitly meant to mean 10, 11, 12, 13, 14, or 15 in length.

The summary and the claims mention antigen precursors and antigens. Asused in the summary and in the claims, a precursor is substantially thefull-length protein encoded by the coding region of the isolated DNA andthe antigen is a peptide which complexes with MHC, preferably HLA, andwhich participates in the immune response as part of that complex. Suchantigens are typically 9 amino acids long, although this may varyslightly.

As used herein, a subject is a human, non-human primate, cow, horse,pig, sheep, goat, dog, cat or rodent. In all embodiments human cancerantigens and human subjects are preferred.

The present invention in one aspect involves the cloning of cDNAsencoding human small cell lung cancer associated antigen precursorsusing autologous antisera of subjects having cancer. The sequences ofthe clones representing genes identified according to the methodsdescribed herein are presented in the attached Sequence Listing. Of theforegoing, it can be seen that some of the clones are novel but may havesome homology to sequences deposited in databases (mainly ESTsequences). Nevertheless, the entire gene sequence was not previouslyknown. In some cases no function was suspected and in other cases, evenif a function was suspected, it was not know that the gene wasassociated with cancer. In all cases, it was not known or suspected thatthe gene encoded a cancer antigen which reacted with antibody fromautologous sera. Analysis of the clone sequences by comparison tonucleic acid and protein databases determined that still other of theclones surprisingly are closely related to other previously-clonedgenes. The sequences of these related genes is also presented in theSequence Listing. The nature of the foregoing genes as encoding antigensrecognized by the immune systems of cancer patients is, of course,unexpected.

The invention thus involves in one aspect cancer associated antigenpolypeptides, genes encoding those polypeptides, functionalmodifications and variants of the foregoing, useful fragments of theforegoing, as well as diagnostics and therapeutics relating thereto.

Homologs and alleles of the cancer associated antigen nucleic acids ofthe invention can be identified by conventional techniques. Thus, anaspect of the invention is those nucleic acid sequences which code forcancer associated antigen precursors. Because this application containsso many sequences, the following chart is provided to identify thevarious groups of sequences discussed in the claims and in the summary:

Nucleic Acid Sequences

-   NA Group 1.    -   (a) nucleic acid molecules which hybridize under stringent        conditions to a molecule consisting of a nucleic acid sequence        selected from the group consisting of SEQ ID NOs: 3-17 and which        code for a cancer associated antigen precursor,    -   (b) deletions, additions and substitutions which code for a        respective cancer associated antigen precursor,    -   (c) nucleic acid molecules that differ from the nucleic acid        molecules of (a) or (b) in codon sequence due to the degeneracy        of the genetic code, and    -   (d) complements of (a), (b) or (c).-   NA Group 2. Fragments of NA Group 1, which code for a polypeptide    which, or a portion of which, binds a MHC molecule to form a complex    recognized by an autologous antibody or lymphocyte.-   NA Group 3. The subset of NA Group 1 where the nucleotide sequence    is selected from the group consisting of:    -   (a) previously unknown human nucleic acids coding for a human        cancer associated antigen precursor set forth as SEQ ID NO:17,    -   (b) deletions, additions and substitutions which code for a        respective human cancer associated antigen precursor,    -   (c) nucleic acid molecules that differ from the nucleic acid        molecules of (a) or (b) in codon sequence due to the degeneracy        of the genetic code, and    -   (d) complements of (a), (b) or (c).-   NA Group 4. Fragments of NA Group 3, which code for a polypeptide    which, or a portion of which, binds to a MHC molecule to form a    complex recognized by an autologous antibody or lymphocyte.-   NA Group 5. A subset of NA Group 1, comprising human cancer    associated antigens that react with allogeneic cancer antisera.

Polypeptide Sequences

-   PP Group 1. Polypeptides encoded by NA Group 1.-   PP Group 2. Polypeptides encoded by NA Group 2-   PP Group 3. Polypeptides encoded by NA Group 3.-   PP Group 4. Polypeptides encoded by NA Group 4.-   PP Group 5. Polypeptides encoded by NA Group 5.

The term “stringent conditions” as used herein refers to parameters withwhich the art is familiar. Nucleic acid hybridization parameters may befound in references which compile such methods, e.g. Molecular Cloning:A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. More specifically, stringentconditions, as used herein, refers, for example, to hybridization at 65°C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH₂PO₄(pH7), 0.5% SDS,2 mM EDTA). SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDSis sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid.After hybridization, the membrane upon which the DNA is transferred iswashed, for example, in 2×SSC at room temperature and then at0.1-0.5×SSC/0.1×SDS at temperatures up to 68° C.

There are other conditions, reagents, and so forth which can be used,which result in a similar degree of stringency. The skilled artisan willbe familiar with such conditions, and thus they are not given here. Itwill be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of cancer associated antigen nucleic acids ofthe invention (e.g., by using lower stringency conditions). The skilledartisan also is familiar with the methodology for screening cells andlibraries for expression of such molecules which then are routinelyisolated, followed by isolation of the pertinent nucleic acid moleculeand sequencing.

In general homologs and alleles typically will share at least 75%nucleotide identity and/or at least 90% amino acid identity to thesequences of cancer associated antigen nucleic acid and polypeptides,respectively, in some instances will share at least 90% nucleotideidentity and/or at least 95% amino acid identity and in still otherinstances will share at least 95% nucleotide identity and/or at least99% amino acid identity. The homology can be calculated using various,publicly available software tools developed by NCBI (Bethesda, Md.) thatcan be obtained through the internet (ftp:/ncbi.nlm.nih.gov/pub/).Exemplary tools include the BLAST system available athttp://www.ncbi.nlm.nih.gov, using default settings. Pairwise andClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittlehydropathic analysis can be obtained using the MacVector sequenceanalysis software (Oxford Molecular Group). Watson-Crick complements ofthe foregoing nucleic acids also are embraced by the invention.

In screening for cancer associated antigen genes, a Southern blot may beperformed using the foregoing conditions, together with a radioactiveprobe. After washing the membrane to which the DNA is finallytransferred, the membrane can be placed against X-ray film to detect theradioactive signal. In screening for the expression of cancer associatedantigen nucleic acids, Northern blot hybridizations using the foregoingconditions (see also the Examples) can be performed on samples takenfrom breast cancer patients or subjects suspected of having a conditioncharacterized by expression of breast cancer associated antigen genes.Amplification protocols such as polymerase chain reaction using primerswhich hybridize to the sequences presented also can be used fordetection of the cancer associated antigen genes or expression thereof.

The small cell lung cancer associated genes correspond to SEQ ID NOs.3-17. The preferred cancer associated antigens for the methods ofdiagnosis disclosed herein are those which were found to react withallogeneic cancer antisera (i.e. NA Group 5). Especially preferred arethe ZIC2 and SOX Group B sequences (SEQ ID Nos: 3, 4, 5, 11 and 12).Encoded polypeptides (e.g., SEQ ID NOS:18-22), peptides and antiserathereto are also preferred for diagnosis.

The invention also includes degenerate nucleic acids which includealternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue into an elongating breast cancerassociated antigen polypeptide. Similarly, nucleotide sequence tripletswhich encode other amino acid residues include, but are not limited to:CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG(arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT(asparagine codons); and ATA, ATC and ATT (isoleucine codons). Otheramino acid residues may be encoded similarly by multiple nucleotidesequences. Thus, the invention embraces degenerate nucleic acids thatdiffer from the biologically isolated nucleic acids in codon sequencedue to the degeneracy of the genetic code.

The invention also provides modified nucleic acid molecules whichinclude additions, substitutions and deletions of one or morenucleotides. In preferred embodiments, these modified nucleic acidmolecules and/or the polypeptides they encode retain at least oneactivity or function of the unmodified nucleic acid molecule and/or thepolypeptides, such as antigenicity, enzymatic activity, receptorbinding, formation of complexes by binding of peptides by MHC class Iand class II molecules, etc. In certain embodiments, the modifiednucleic acid molecules encode modified polypeptides, preferablypolypeptides having conservative amino acid substitutions as aredescribed elsewhere herein. The modified nucleic acid molecules arestructurally related to the unmodified nucleic acid molecules and inpreferred embodiments are sufficiently structurally related to theunmodified nucleic acid molecules so that the modified and unmodifiednucleic acid molecules hybridize under stringent conditions known to oneof skill in the art.

For example, modified nucleic acid molecules which encode polypeptideshaving single amino acid changes can be prepared. Each of these nucleicacid molecules can have one, two or three nucleotide substitutionsexclusive of nucleotide changes corresponding to the degeneracy of thegenetic code as described herein. Likewise, modified nucleic acidmolecules which encode polypeptides having two amino acid changes can beprepared which have, e.g., 2-6 nucleotide changes. Numerous modifiednucleic acid molecules like these will be readily envisioned by one ofskill in the art, including for example, substitutions of nucleotides incodons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2 and 6, and soon. In the foregoing example, each combination of two amino acids isincluded in the set of modified nucleic acid molecules, as well as allnucleotide substitutions which code for the amino acid substitutions.Additional nucleic acid molecules that encode polypeptides havingadditional substitutions (i.e., 3 or more), additions or deletions(e.g., by introduction of a stop codon or a splice site(s)) also can beprepared and are embraced by the invention as readily envisioned by oneof ordinary skill in the art. Any of the foregoing nucleic acids orpolypeptides can be tested by routine experimentation for retention ofstructural relation or activity to the nucleic acids and/or polypeptidesdisclosed herein.

The invention also provides isolated unique fragments of cancerassociated antigen nucleic acid sequences or complements thereof. Aunique fragment is one that is a ‘signature’ for the larger nucleicacid. It, for example, is long enough to assure that its precisesequence is not found in molecules within the human genome outside ofthe cancer associated antigen nucleic acids defined above (and humanalleles). Those of ordinary skill in the art may apply no more thanroutine procedures to determine if a fragment is unique within the humangenome. Unique fragments, however, exclude fragments completely composedof the nucleotide sequences of any of GenBank accession numbers listedin Table 4 or other previously published sequences as of the filing dateof the priority documents for sequences listed in a respective prioritydocument or the filing date of this application for sequences listed forthe first time in this application which overlap the sequences of theinvention.

A fragment which is completely composed of the sequence described in theforegoing GenBank deposits is one which does not include any of thenucleotides unique to the sequences of the invention. Thus, a uniquefragment must contain a nucleotide sequence other than the exactsequence of those in GenBank or fragments thereof. The difference may bean addition, deletion or substitution with respect to the GenBanksequence or it may be a sequence wholly separate from the GenBanksequence.

Unique fragments can be used as probes in Southern and Northern blotassays to identify such nucleic acids, or can be used in amplificationassays such as those employing PCR. As known to those skilled in theart, large probes such as 200, 250, 300 or more nucleotides arepreferred for certain uses such as Southern and Northern blots, whilesmaller fragments will be preferred for uses such as PCR. Uniquefragments also can be used to produce fusion proteins for generatingantibodies or determining binding of the polypeptide fragments, or forgenerating immunoassay components. Likewise, unique fragments can beemployed to produce nonfused fragments of the cancer associated antigenpolypeptides, useful, for example, in the preparation of antibodies, andin immunoassays. Unique fragments further can be used as antisensemolecules to inhibit the expression of cancer associated antigen nucleicacids and polypeptides, particularly for therapeutic purposes asdescribed in greater detail below.

As will be recognized by those skilled in the art, the size of theunique fragment will depend upon its conservancy in the genetic code.Thus, some regions of cancer associated antigen sequences andcomplements thereof will require longer segments to be unique whileothers will require only short segments, typically between 12 and 32nucleotides (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31 and 32 or more bases long, up to the entirelength of the disclosed sequence. As mentioned above, this disclosureintends to embrace each and every fragment of each sequence, beginningat the first nucleotide, the second nucleotide and so on, up to 8nucleotides short of the end, and ending anywhere from nucleotide number8, 9, 10 and so on for each sequence, up to the very last nucleotide(provided the sequence is unique as described above).

Virtually any segment of the polypeptide coding region of novel cancerassociated antigen nucleic acids, or complements thereof, that is 18 ormore nucleotides in length will be unique. Those skilled in the art arewell versed in methods for selecting such sequences, typically on thebasis of the ability of the unique fragment to selectively distinguishthe sequence of interest from other sequences in the human genome of thefragment to those on known databases typically is all that is necessary,although in vitro confirmatory hybridization and sequencing analysis maybe performed.

Especially preferred include nucleic acids encoding a series ofepitopes, known as “polytopes”. The epitopes can be arranged insequential or overlapping fashion (see, e.g., Thomson et al., Proc.Natl. Acad. Sci. USA 92:5845-5849, 1995; Gilbert et al., NatureBiotechnol. 15:1280-1284, 1997), with or without the natural flankingsequences, and can be separated by unrelated linker sequences ifdesired. The polytope is processed to generate individual epitopes whichare recognized by the immune system for generation of immune responses.

Thus, for example, peptides derived from a polypeptide having an aminoacid sequence encoded by one of the nucleic acid disclosed herein, andwhich are presented by MHC molecules and recognized by CTL or T helperlymphocytes, can be combined with peptides from one or more other cancerassociated antigens (e.g. by preparation of hybrid nucleic acids orpolypeptides) to form “polytopes”. The two or more peptides (or nucleicacids encoding the peptides) can be selected from those describedherein, or they can include one or more peptides of previously knowncancer associated antigens. Exemplary cancer associated peptide antigensthat can be administered to induce or enhance an immune response arederived from tumor associated genes and encoded proteins includingMAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8,MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, BAGE-1, RAGE-1, LB33/MUM-1,PRAME, NAG, MAGE-B2, MAGE-B3, MAGE-B4, tyrosinase, brain glycogenphosphorylase, Melan-A, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5,NY-ESO-1, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-4, SSX-5, SCP-1 andCT-7. See, for example, PCT application publication no. W096/10577.Other examples will be known to one of ordinary skill in the art and canbe used in the invention in a like manner as those disclosed herein.

Other examples of HLA class I and HLA class II binding peptides will beknown to one of ordinary skill in the art. For example, see thefollowing references: Coulie, Stem Cells 13:393-403, 1995; Traversari etal., J. Exp. Med. 176:1453-1457, 1992; Chaux et al., J. Immunol.163:2928-2936, 1999; Fujie et al., Int. J. Cancer 80:169-172, 1999;Tanzarella et al., Cancer Res. 59:2668-2674, 1999; van der Bruggen etal., Eur. J. Immunol. 24:2134-2140, 1994; Chaux et al., J. Exp. Med.189:767-778, 1999; Kawashima et al, Hum. Immunol. 59:1-14, 1998; Taharaet al., Clin. Cancer Res. 5:2236-2241, 1999; Gaugler et al., J. Exp.Med. 179:921-930, 1994; van der Bruggen et al., Eur. J. Immunol.24:3038-3043, 1994; Tanaka et al., Cancer Res. 57:4465-4468, 1997; Oisoet al., Int. J. Cancer 81:387-394, 1999; Herman et al., Immunogenetics43:377-383, 1996; Manici et al., J. Exp. Med. 189:871-876, 1999; Duffouret al., Eur. J. Immunol. 29:3329-3337, 1999; Zom et al., Eur. J.Immunol. 29:602-607, 1999; Huang et al., J. Immunol. 162:6849-6854,1999; Boël et al., Immunity 2:167-175, 1995; Van den Eynde et al., J.Exp. Med. 182:689-698, 1995; De Backer et al., Cancer Res. 59:3157-3165,1999; Jager et al., J. Exp. Med. 187:265-270, 1998; Wang et al., J.Immunol. 161:3596-3606, 1998; Aamoudse et al., Int. J. Cancer82:442-448, 1999; Guilloux et al., J. Exp. Med. 183:1173-1183, 1996;Lupetti et al., J. Exp. Med. 188:1005-1016, 1998; Wölfel et al., Eur. J.Immunol. 24:759-764, 1994; Skipper et al., J. Exp. Med. 183:527-534,1996; Kang et al., J. Immunol. 155:1343-1348, 1995; Morel et al., Int.J. Cancer 83:755-759, 1999; Brichard et al., Eur. J. Immunol.26:224-230, 1996; Kittlesen et al., J. Immunol. 160:2099-2106, 1998;Kawakami et al., J. Immunol. 161:6985-6992, 1998; Topalian et al., J.Exp. Med. 183:1965-1971, 1996; Kobayashi et al., Cancer Research58:296-301, 1998; Kawakami et al., J. Immunol. 154:3961-3968, 1995; Tsaiet al., J. Immunol. 158:1796-1802, 1997; Cox et al., Science264:716-719, 1994; Kawakami et al., Proc. Natl. Acad. Sci. USA91:6458-6462, 1994; Skipper et al., J. Immunol. 157:5027-5033, 1996;Robbins et al., J. Immunol. 159:303-308, 1997; Castelli et al, J.Immunol. 162:1739-1748, 1999; Kawakami et al., J. Exp. Med. 180:347-352,1994; Castelli et al., J. Exp. Med. 181:363-368, 1995; Schneider et al.,Int. J. Cancer 75:451-458, 1998; Wang et al., J. Exp. Med.183:1131-1140, 1996; Wang et al., J. Exp. Med. 184:2207-2216, 1996;Parkhurst et al., Cancer Research 58:4895-4901, 1998; Tsang et al., J.Natl Cancer Inst 87:982-990, 1995; Correale et al., J Natl Cancer Inst89:293-300, 1997; Coulie et al., Proc. Natl. Acad. Sci. USA92:7976-7980, 1995; Wölfel et al., Science 269:1281-1284, 1995; Robbinset al., J. Exp. Med. 183:1185-1192, 1996; Brandle et al., J. Exp. Med.183:2501-2508, 1996; ten Bosch et al., Blood 88:3522-3527, 1996;Mandruzzato et al., J. Exp. Med. 186:785-793, 1997; Gueguen et al., J.Immunol. 160:6188-6194, 1998; Gjertsen et al., Int. J. Cancer72:784-790, 1997; Gaudin et al., J. Immunol. 162:1730-1738, 1999; Chiariet al., Cancer Res. 59:5785-5792, 1999; Hogan et al., Cancer Res.58:5144-5150, 1998; Pieper et al., J. Exp. Med. 189:757-765, 1999; Wanget al., Science 284:1351-1354, 1999; Fisk et al., J. Exp. Med.181:2109-2117, 1995; Brossart et al., Cancer Res. 58:732-736, 1998;Ropke et al., Proc. Natl. Acad. Sci. USA 93:14704-14707, 1996; Ikeda etal., Immunity 6:199-208, 1997; Ronsin et al., J. Immunol. 163:483-490,1999; Vonderheide et al., Immunity 10:673-679,1999.

One of ordinary skill in the art can prepare polypeptides comprising oneor more peptides and one or more of the foregoing cancer associatedpeptides, or nucleic acids encoding such polypeptides, according tostandard procedures of molecular biology.

Thus polytopes are groups of two or more potentially immunogenic orimmune response stimulating peptides which can be joined together invarious arrangements (e.g. concatenated, overlapping). The polytope (ornucleic acid encoding the polytope) can be administered in a standardimmunization protocol, e.g. to animals, to test the effectiveness of thepolytope in stimulating, enhancing and/or provoking an immune response.

The peptides can be joined together directly or via the use of flankingsequences to form polytopes, and the use of polytopes as vaccines iswell known in the art (see, e.g., Thomson et al., Proc. Acad. Natl.Acad. Sci USA 92(13):5845-5849, 1995; Gilbert et al., Nature Biotechnol.15(12):1280-1284, 1997; Thomson et al., J. Immunol. 157(2):822-826,1996; Tam et al., J. Exp. Med. 171(1):299-306, 1990). For example, Tamshowed that polytopes consisting of both MHC class I and class IIbinding epitopes successfully generated antibody and protective immunityin a mouse model. Tam also demonstrated that polytopes comprising“strings” of epitopes are processed to yield individual epitopes whichare presented by MHC molecules and recognized by CTLs. Thus polytopescontaining various numbers and combinations of epitopes can be preparedand tested for recognition by CTLs and for efficacy in increasing animmune response.

It is known that tumors express a set of tumor antigens, of which onlycertain subsets may be expressed in the tumor of any given patient.Polytopes can be prepared which correspond to the different combinationof epitopes representing the subset of tumor rejection antigensexpressed in a particular patient. Polytopes also can be prepared toreflect a broader spectrum of tumor rejection antigens known to beexpressed by a tumor type. Polytopes can be introduced to a patient inneed of such treatment as polypeptide structures, or via the use ofnucleic acid delivery systems known in the art (see, e.g., Allsopp etal., Eur. J. Immunol. 26(8):1951-1959, 1996). Adenovirus, pox virus,Ty-virus like particles, adeno-associated virus, plasmids, bacteria,etc. can be used in such delivery. One can test the polytope deliverysystems in mouse models to determine efficacy of the delivery system.The systems also can be tested in human clinical trials.

In instances in which a human HLA class I molecule presents tumorrejection antigens derived from cancer associated nucleic acids, theexpression vector may also include a nucleic acid sequence coding forthe HLA molecule that presents any particular tumor rejection antigenderived from these nucleic acids and polypeptides. Alternatively, thenucleic acid sequence coding for such a HLA molecule can be containedwithin a separate expression vector. In a situation where the vectorcontains both coding sequences, the single vector can be used totransfect a cell which does not normally express either one. Where thecoding sequences for a cancer associated antigen precursor and the HLAmolecule which presents it are contained on separate expression vectors,the expression vectors can be cotransfected. The cancer associatedantigen precursor coding sequence may be used alone, when, e.g. the hostcell already expresses a HLA molecule which presents a cancer associatedantigen derived from precursor molecules. Of course, there is no limiton the particular host cell which can be used. As the vectors whichcontain the two coding sequences may be used in any antigen-presentingcells if desired, and the gene for cancer associated antigen precursorcan be used in host cells which do not express a HLA molecule whichpresents a cancer associated antigen. Further, cell-free transcriptionsystems may be used in lieu of cells.

As mentioned above, the invention embraces antisense oligonucleotidesthat selectively bind to a nucleic acid molecule encoding a cancerassociated antigen polypeptide, to reduce the expression of cancerassociated antigens. This is desirable in virtually any medicalcondition wherein a reduction of expression of cancer associatedantigens is desirable, e.g., in the treatment of cancer. This is alsouseful for in vitro or in vivo testing of the effects of a reduction ofexpression of one or more cancer associated antigens.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that MRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene or transcript. Those skilled in theart will recognize that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence. It ispreferred that the antisense oligonucleotide be constructed and arrangedso as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon the sequences of nucleic acids encoding breastcancer associated antigen, or upon allelic or homologous genomic and/orcDNA sequences, one of skill in the art can easily choose and synthesizeany of a number of appropriate antisense molecules for use in accordancewith the present invention. In order to be sufficiently selective andpotent for inhibition, such antisense oligonucleotides should compriseat least 10 and, more preferably, at least 15 consecutive bases whichare complementary to the target, although in certain cases modifiedoligonucleotides as short as 7 bases in length have been usedsuccessfully as antisense oligonucleotides (Wagner et al., NatureBiotechnol. 14:840-844, 1996). Most preferably, the antisenseoligonucleotides comprise a complementary sequence of 20-30 bases.Although oligonucleotides may be chosen which are antisense to anyregion of the gene or MRNA transcripts, in preferred embodiments theantisense oligonucleotides correspond to N-terminal or 5′ upstream sitessuch as translation initiation, transcription initiation or promotersites. In addition, 3′-untranslated regions may be targeted. Targetingto MRNA splicing sites has also been used in the art but may be lesspreferred if alternative mRNA splicing occurs. In addition, theantisense is targeted, preferably, to sites in which mRNA secondarystructure is not expected (see, e.g., Sainio et al., Cell Mol.Neurobiol. 14(5):439-457, 1994) and at which proteins are not expectedto bind. Finally, although the listed sequences are cDNA sequences, oneof ordinary skill in the art may easily derive the genomic DNAcorresponding to the cDNA of a cancer associated antigen. Thus, thepresent invention also provides for antisense oligonucleotides which arecomplementary to the genomic DNA corresponding to nucleic acids encodingcancer associated antigens. Similarly, antisense to allelic orhomologous cDNAs and genomic DNAs are enabled without undueexperimentation.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic intemucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose. Base analogs such asC-5 propyne modified bases also can be included (Nature Biotechnol.14:840-844, 1996). The present invention, thus, contemplatespharmaceutical preparations containing modified antisense molecules thatare complementary to and hybridizable with, under physiologicalconditions, nucleic acids encoding the cancer associated antigenpolypeptides, together with pharmaceutically acceptable carriers.

Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art. The compositions should be sterile and contain atherapeutically effective amount of the antisense oligonucleotides in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers., and other materials which are wellknown in the art, as further described below.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmids,phagemids and virus genomes. A cloning vector is one which is able toreplicate autonomously or integrated in the genone in a host cell, andwhich is further characterized by one or more endonuclease restrictionsites at which the vector may be cut in a determinable fashion and intowhich a desired DNA sequence may be ligated such that the newrecombinant vector retains its ability to replicate in the host cell. Inthe case of plasmids, replication of the desired sequence may occur manytimes as the plasmid increases in copy number within the host bacteriumor just a single time per host before the host reproduces by mitosis. Inthe case of phage, replication may occur actively during a lytic phaseor passively during a lysogenic phase. An expression vector is one intowhich a desired DNA sequence may be inserted by restriction and ligationsuch that it is operably joined to regulatory sequences and may beexpressed as an RNA transcript. Vectors may further contain one or moremarker sequences suitable for use in the identification of cells whichhave or have not been transformed or transfected with the vector.Markers include, for example, genes encoding proteins which increase ordecrease either resistance or sensitivity to antibiotics or othercompounds, genes which encode enzymes whose activities are detectable bystandard assays known in the art (e.g., β-galactosidase, luciferase oralkaline phosphatase), and genes which visibly affect the phenotype oftransformed or transfected cells, hosts, colonies or plaques (e.g.,green fluorescent protein). Preferred vectors are those capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribed regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding a breast cancer associated antigenpolypeptide or fragment or variant thereof. That heterologous DNA (RNA)is placed under operable control of transcriptional elements to permitthe expression of the heterologous DNA in the host cell.

Preferred systems for MRNA expression in mammalian cells are those suchas pRc/CMV (available from Invitrogen, Carlsbad, CA) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen), which contains an Epstein Barr Virus (EBV) originof replication, facilitating the maintenance of plasmid as a multicopyextrachromosomal element. Another expression vector is the pEF-BOSplasmid containing the promoter of polypeptide Elongation Factor 1α,which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992). The use of the adenovirus as an Adeno.P1A recombinant for theexpression of an antigen is disclosed by Wamier et al., in intradermalinjection in mice for immunization against PIA (Int. J. Cancer,67:303-310, 1996). Additional vectors for delivery of nucleic acid areprovided below.

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of a vector and oneor more of the previously discussed cancer associated antigen nucleicacid molecules. Other components may be added, as desired, as long asthe previously mentioned nucleic acid molecules, which are required, areincluded. The invention also includes kits for amplification of a cancerassociated antigen nucleic acid, including at least one pair ofamplification primers which hybridize to a cancer associated antigennucleic acid. The primers preferably are 12-32 nucleotides in length andare non-overlapping to prevent formation of “primer-dimers”. One of theprimers will hybridize to one strand of the cancer associated antigennucleic acid and the second primer will hybridize to the complementarystrand of the cancer associated antigen nucleic acid, in an arrangementwhich permits amplification of the cancer associated antigen nucleicacid. Selection of appropriate primer pairs is standard in the art. Forexample, the selection can be made with assistance of a computer programdesigned for such a purpose, optionally followed by testing the primersfor amplification specificity and efficiency.

The invention also permits the construction of cancer associated antigengene “knock-outs” in cells and in animals, providing materials forstudying certain aspects of cancer and immune system responses tocancer.

The invention also provides isolated polypeptides (including wholeproteins and partial proteins) encoded by the foregoing cancerassociated antigen nucleic acids. Such polypeptides are useful, forexample, alone or as fusion proteins to generate antibodies, ascomponents of an immunoassay or diagnostic assay or as therapeutics.Cancer associated antigen polypeptides can be isolated from biologicalsamples including tissue or cell homogenates, and can also be expressedrecombinantly in a variety of prokaryotic and eukaryotic expressionsystems by constructing an expression vector appropriate to theexpression system, introducing the expression vector into the expressionsystem, and isolating the recombinantly expressed protein. Shortpolypeptides, including antigenic peptides (such as are presented by MHCmolecules on the surface of a cell for immune recognition) also can besynthesized chemically using well-established methods of peptidesynthesis.

A unique fragment of a cancer associated antigen polypeptide, ingeneral, has the features and characteristics of unique fragments asdiscussed above in connection with nucleic acids. As will be recognizedby those skilled in the art, the size of the unique fragment will dependupon factors such as whether the fragment constitutes a portion of aconserved protein domain. Thus, some regions of cancer associatedantigens will require longer segments to be unique while others willrequire only short segments, typically between 5 and 12 amino acids(e.g. 5, 6, 7, 8, 9, 10, 11 or 12 or more amino acids including eachinteger up to the full length).

Unique fragments of a polypeptide preferably are those fragments whichretain a distinct functional capability of the polypeptide. Functionalcapabilities which can be retained in a unique fragment of a polypeptideinclude interaction with antibodies, interaction with other polypeptidesor fragments thereof, selective binding of nucleic acids or proteins,and enzymatic activity. One important activity is the ability to act asa signature for identifying the polypeptide. Another is the ability tocomplex with HLA and to provoke in a human an immune response. Thoseskilled in the art are well versed in methods for selecting unique aminoacid sequences, typically on the basis of the ability of the uniquefragment to selectively distinguish the sequence of interest fromnon-family members. A comparison of the sequence of the fragment tothose on known databases typically is all that is necessary.

The invention embraces variants of the cancer associated antigenpolypeptides described above. As used herein, a “variant” of a cancerassociated antigen polypeptide is a polypeptide which contains one ormore modifications to the primary amino acid sequence of a cancerassociated antigen polypeptide. Modifications which create a cancerassociated antigen variant can be made to a cancer associated antigenpolypeptide 1) to reduce or eliminate an activity of a cancer associatedantigen polypeptide; 2) to enhance a property of a cancer associatedantigen polypeptide, such as protein stability in an expression systemor the stability of protein-protein binding; 3) to provide a novelactivity or property to a cancer associated antigen polypeptide, such asaddition of an antigenic epitope or addition of a detectable moiety; or4) to provide equivalent or better binding to an HLA molecule.Modifications to a cancer associated antigen polypeptide are typicallymade to the nucleic acid which encodes the cancer associated antigenpolypeptide, and can include deletions, point mutations, truncations,amino acid substitutions and additions of amino acids or non-amino acidmoieties. Alternatively, modifications can be made directly to thepolypeptide, such as by cleavage, addition of a linker molecule,addition of a detectable moiety, such as biotin, addition of a fattyacid, and the like. Modifications also embrace fusion proteinscomprising all or part of the cancer associated antigen amino acidsequence. One of skill in the art will be familiar with methods forpredicting the effect on protein conformation of a change in proteinsequence, and can thus “design” a variant cancer associated antigenpolypeptide according to known methods. One example of such a method isdescribed by Dahiyat and Mayo in Science 278:82-87, 1997, wherebyproteins can be designed de novo. The method can be applied to a knownprotein to vary a only a portion of the polypeptide sequence. Byapplying the computational methods of Dahiyat and Mayo, specificvariants of a cancer associated antigen polypeptide can be proposed andtested to determine whether the variant retains a desired conformation.

In general, variants include cancer associated antigen polypeptideswhich are modified specifically to alter a feature of the polypeptideunrelated to its desired physiological activity. For example, cysteineresidues can be substituted or deleted to prevent unwanted disulfidelinkages. Similarly, certain amino acids can be changed to enhanceexpression of a breast cancer associated antigen polypeptide byeliminating proteolysis by proteases in an expression system (e.g.,dibasic amino acid residues in yeast expression systems in which KEX2protease activity is present).

Mutations of a nucleic acid which encode a cancer associated antigenpolypeptide preferably preserve the amino acid reading frame of thecoding sequence, and preferably do not create regions in the nucleicacid which are likely to hybridize to form secondary structures, such ahairpins or loops, which can be deleterious to expression of the variantpolypeptide.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with the desired properties. Further mutations can be madeto variants (or to non-variant cancer associated antigen polypeptides)which are silent as to the amino acid sequence of the polypeptide, butwhich provide preferred codons for translation in a particular host. Thepreferred codons for translation of a nucleic acid in, e.g., E. coli,are well known to those of ordinary skill in the art. Still othermutations can be made to the noncoding sequences of a cancer associatedantigen gene or cDNA clone to enhance expression of the polypeptide. Theactivity of variants of cancer associated antigen polypeptides can betested by cloning the gene encoding the variant cancer associatedantigen polypeptide into a bacterial or mammalian expression vector,introducing the vector into an appropriate host cell, expressing thevariant cancer associated antigen polypeptide, and testing for afunctional capability of the cancer associated antigen polypeptides asdisclosed herein. For example, the variant cancer associated antigenpolypeptide can be tested for reaction with autologous or allogeneicsera as disclosed in the Examples. Preparation of other variantpolypeptides may favor testing of other activities, as will be known toone of ordinary skill in the art.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in cancer associated antigen polypeptides toprovide functionally equivalent variants of the foregoing polypeptides,i.e, the variants retain the functional capabilities of the cancerassociated antigen polypeptides. As used herein, a “conservative aminoacid substitution” refers to an amino acid substitution which does notalter the relative charge or size characteristics of the protein inwhich the amino acid substitution is made. Variants can be preparedaccording to methods for altering polypeptide sequence known to one ofordinary skill in the art such as are found in references which compilesuch methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook,et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Exemplaryfunctionally equivalent variants of the cancer associated antigenpolypeptides include conservative amino acid substitutions of in theamino acid sequences of proteins disclosed herein. Conservativesubstitutions of amino acids include substitutions made amongst aminoacids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K,R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

For example, upon determining that a peptide derived from a cancerassociated antigen polypeptide is presented by an MHC molecule andrecognized by CTLs (e.g., as described in the Examples), one can makeconservative amino acid substitutions to the amino acid sequence of thepeptide, particularly at residues which are thought not to be directcontact points with the MHC molecule. For example, methods foridentifying functional variants of HLA class II binding peptides areprovided in a published PCT application of Strominger and Wucherpfennig(PCT/US96/03182). Peptides bearing one or more amino acid substitutionsalso can be tested for concordance with known HLA/MHC motifs prior tosynthesis using, e.g. the computer program described by D'Amaro andDrijfhout (D'Amaro et al., Human Immunol. 43:13-18, 1995; Drijfhout etal., Human Immunol. 43:1-12, 1995). The substituted peptides can then betested for binding to the MHC molecule and recognition by CTLs whenbound to MHC. These variants can be tested for improved stability andare useful, inter alia, in vaccine compositions.

Conservative amino-acid substitutions in the amino acid sequence ofcancer associated antigen polypeptides to produce functionallyequivalent variants of cancer associated antigen polypeptides typicallyare made by alteration of a nucleic acid encoding a cancer associatedantigen polypeptide. Such substitutions can be made by a variety ofmethods known to one of ordinary skill in the art. For example, aminoacid substitutions may be made by PCR-directed mutation, site-directedmutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad.Sci. U.S.A. 82: 488-492, 1985), or by chemical synthesis of a geneencoding a cancer associated antigen polypeptide. Where amino acidsubstitutions are made to a small unique fragment of a cancer associatedantigen polypeptide, such as an antigenic epitope recognized byautologous or allogeneic sera or cytolytic T lymphocytes, thesubstitutions can be made by directly synthesizing the peptide. Theactivity of functionally equivalent fragments of cancer associatedantigen polypeptides can be tested by cloning the gene encoding thealtered cancer associated antigen polypeptide into a bacterial ormammalian expression vector, introducing the vector into an appropriatehost cell, expressing the altered cancer associated antigen polypeptide,and testing for a functional capability of the cancer associated antigenpolypeptides as disclosed herein. Peptides which are chemicallysynthesized can be tested directly for function, e.g., for binding toantisera recognizing associated antigens.

The invention as described herein has a number of uses, some of whichare described elsewhere herein. First, the invention permits isolationof the cancer associated antigen protein molecules. A variety ofmethodologies well-known to the skilled practitioner can be utilized toobtain isolated cancer associated antigen molecules. The polypeptide maybe purified from cells which naturally produce the polypeptide bychromatographic means or immunological recognition. Alternatively, anexpression vector may be introduced into cells to cause production ofthe polypeptide. In another method, mRNA transcripts may bemicroinjected or otherwise introduced into cells to cause production ofthe encoded polypeptide. Translation of mRNA in cell-free extracts suchas the reticulocyte lysate system also may be used to producepolypeptide. Those skilled in the art also can readily follow knownmethods for isolating cancer associated antigen polypeptides. Theseinclude, but are not limited to, immunochromatography, HPLC,size-exclusion chromatography, ion-exchange chromatography andimmune-affinity chromatography.

The isolation and identification of cancer associated antigen genes alsomakes it possible for the artisan to diagnose a disorder characterizedby expression of cancer associated antigens. These methods involvedetermining expression of one or more cancer associated antigen nucleicacids, and/or encoded cancer associated antigen polypeptides and/orpeptides derived therefrom. In the former situation, such determinationscan be carried out via any standard nucleic acid determination assay,including the polymerase chain reaction, or assaying with labeledhybridization probes. In the latter situation, such determinations canbe carried out by screening patient antisera for recognition of thepolypeptide.

The invention also makes it possible isolate proteins which bind tocancer associated antigens as disclosed herein, including antibodies andcellular binding partners of the cancer associated antigens. Additionaluses are described further herein.

The invention also provides, in certain embodiments, “dominant negative”polypeptides derived from cancer associated antigen polypeptides. Adominant negative polypeptide is an inactive variant of a protein,which, by interacting with the cellular machinery, displaces an activeprotein from its interaction with the cellular machinery or competeswith the active protein, thereby reducing the effect of the activeprotein. For example, a dominant negative receptor which binds a ligandbut does not transmit a signal in response to binding of the ligand canreduce the biological effect of expression of the ligand. Likewise, adominant negative catalytically-inactive kinase which interacts normallywith target proteins but does not phosphorylate the target proteins canreduce phosphorylation of the target proteins in response to a cellularsignal. Similarly, a dominant negative transcription factor which bindsto a promoter site in the control region of a gene but does not increasegene transcription can reduce the effect of a normal transcriptionfactor by occupying promoter binding sites without increasingtranscription.

The end result of the expression of a dominant negative polypeptide in acell is a reduction in function of active proteins. One of ordinaryskill in the art can assess the potential for a dominant negativevariant of a protein, and using standard mutagenesis techniques tocreate one or more dominant negative variant polypeptides. For example,given the teachings contained herein of small cell lung cancerassociated antigens, especially those which are similar to knownproteins which have known activities, one of ordinary skill in the artcan modify the sequence of the cancer associated antigens bysite-specific mutagenesis, scanning mutagenesis, partial gene deletionor truncation, and the like. See, e.g., U.S. Pat. No. 5,580,723 andSambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, 1989. The skilled artisan then cantest the population of mutagenized polypeptides for diminution in aselected and/or for retention of such an activity. Other similar methodsfor creating and testing dominant negative variants of a protein will beapparent to one of ordinary skill in the art.

The invention also involves agents such as polypeptides which bind tocancer associated antigen polypeptides. Such binding agents can be used,for example, in screening assays to detect the presence or absence ofcancer associated antigen polypeptides and complexes of cancerassociated antigen polypeptides and their binding partners and inpurification protocols to isolated cancer associated antigenpolypeptides and complexes of cancer associated antigen polypeptides andtheir binding partners. Such agents also can be used to inhibit thenative activity of the cancer associated antigen polypeptides, forexample, by binding to such polypeptides.

The invention, therefore, embraces peptide binding agents which, forexample, can be antibodies or fragments of antibodies having the abilityto selectively bind to cancer associated antigen polypeptides.Antibodies include polyclonal and monoclonal antibodies, preparedaccording to conventional methodology.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modem lmmunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymnatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. See, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539,5,585,089, 5,693,762 and 5,859,205.

Thus, for example, PCT International Publication Number WO 92/04381teaches the production and use of humanized murine RSV antibodies inwhich at least a portion of the murine FR regions have been replaced byFR regions of human origin. Such antibodies, including fragments ofintact antibodies with antigen-binding ability, are often referred to as“chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to cancer associated antigen polypeptides, andcomplexes of both cancer associated antigen polypeptides and theirbinding partners. These polypeptides may be derived also from sourcesother than antibody technology. For example, such polypeptide bindingagents can be provided by degenerate peptide libraries which can bereadily prepared in solution, in immobilized form or as phage displaylibraries. Combinatorial libraries also can be synthesized of peptidescontaining one or more amino acids. Libraries further can be synthesizedof peptoids and non-peptide synthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind tothe cancer associated antigen polypeptide. This process can be repeatedthrough several cycles of reselection of phage that bind to the cancerassociated antigen polypeptide. Repeated rounds lead to enrichment ofphage bearing particular sequences. DNA sequence analysis can beconducted to identify the sequences of the expressed polypeptides. Theminimal linear portion of the sequence that binds to the cancerassociated antigen polypeptide can be determined. One can repeat theprocedure using a biased library containing inserts containing part orall of the minimal linear portion plus one or more additional degenerateresidues upstream or downstream thereof. Yeast two-hybrid screeningmethods also may be used to identify polypeptides that bind to thecancer associated antigen polypeptides. Thus, the cancer associatedantigen polypeptides of the invention, or a fragment thereof, can beused to screen peptide libraries, including phage display libraries, toidentify and select peptide binding partners of the cancer associatedantigen polypeptides of the invention. Such molecules can be used, asdescribed, for screening assays, for purification protocols, forinterfering directly with the functioning of cancer associated antigenand for other purposes that will be apparent to those of ordinary skillin the art.

As detailed herein, the foregoing antibodies and other binding moleculesmay be used for example to identify tissues expressing protein or topurify protein. Antibodies also may be coupled to specific diagnosticlabeling agents for imaging of cells and tissues that express cancerassociated antigens or to therapeutically useful agents according tostandard coupling procedures. Diagnostic agents include, but are notlimited to, barium sulfate, iocetamic acid, iopanoic acid, ipodatecalcium, diatrizoate sodium, diatrizoate meglumine, metrizamide,tyropanoate sodium and radiodiagnostics including positron emitters suchas fluorine-18 and carbon-11, gamma emitters such as iodine-123,technitium-99m, iodine-131 and indium-111, nuclides for nuclear magneticresonance such as fluorine and gadolinium. Other diagnostic agentsuseful in the invention will be apparent to one of ordinary skill in theart. As used herein, “therapeutically useful agents” include anytherapeutic molecule which desirably is targeted selectively to a cellexpressing one of the cancer antigens disclosed herein, includingantineoplastic agents, radioiodinated compounds, toxins, othercytostatic or cytolytic drugs, and so forth. Antineoplastic therapeuticsare well known and include: aminoglutethimide, azathioprine, bleomycinsulfate, busulfan, carmustine, chlorambucil, cisplatin,cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin,daunorubicin, doxorubicin, taxol, etoposide, fluorouracil, interferon-α,lomustine, mercaptopurine, methotrexate, mitotane, procarbazine HCl,thioguanine, vinblastine sulfate and vincristine sulfate. Additionalantineoplastic agents include those disclosed in Chapter 52,Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and theintroduction thereto, 1202-1263, of Goodman and Gilman's “ThePharmacological Basis of Therapeutics”, Eighth Edition, 1990,McGraw-Hill, Inc. (Health Professions Division). Toxins can be proteinssuch as, for example, pokeweed anti-viral protein, cholera toxin,pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin, orPseudomonas exotoxin. Toxin moieties can also be high energy-emittingradionuclides such as cobalt-60.

In the foregoing methods, antibodies prepared according to the inventionalso preferably are specific for the small cell lung cancer associatedantigen/MHC complexes described herein.

When “disorder” is used herein, it refers to any pathological conditionwhere the cancer associated antigens are expressed. An example of such adisorder is cancer, with lung cancers including small cell lung cancerand non-small cell lung cancer, melanoma, colon cancer, breast cancer,head and neck cancer, transitional cancer, leiomyosarcoma and synovialsarcoma as particular examples.

Samples of tissue and/or cells for use in the various methods describedherein can be obtained through standard methods such as tissue biopsy,including punch biopsy and cell scraping, and collection of blood orother bodily fluids by aspiration or other methods.

In certain embodiments of the invention, an immunoreactive cell sampleis removed from a subject. By “immunoreactive cell” is meant a cellwhich can mature into an immune cell (such as a B cell, a helper T cell,or a cytolytic T cell) upon appropriate stimulation. Thus immunoreactivecells include CD34⁺ hematopoietic stem cells, immature T cells andimmature B cells. When it is desired to produce cytolytic T cells whichrecognize a cancer associated antigen, the immunoreactive cell iscontacted with a cell which expresses a cancer associated antigen underconditions favoring production, differentiation and/or selection ofcytolytic T cells; the differentiation of the T cell precursor into acytolytic T cell upon exposure to antigen is similar to clonal selectionof the immune system.

Some therapeutic approaches based upon the disclosure are premised on aresponse by a subject's immune system, leading to lysis of antigenpresenting cells, such as breast cancer cells which present one or morecancer associated antigens. One such approach is the administration ofautologous CTLs specific to a cancer associated antigen/MHC complex to asubject with abnormal cells of the phenotype at issue. It is within theability of one of ordinary skill in the art to develop such CTLs invitro. An example of a method for T cell differentiation is presented inInternational Application number PCT/US96/05607. Generally, a sample ofcells taken from a subject, such as blood cells, are contacted with acell presenting the complex and capable of provoking CTLs toproliferate. The target cell can be a transfectant, such as a COS cell.These transfectants present the desired complex of their surface and,when combined with a CTL of interest, stimulate its proliferation. COScells are widely available, as are other suitable host cells. Specificproduction of CTL clones is well known in the art. The clonally expandedautologous CTLs then are administered to the subject.

Another method for selecting antigen-specific CTL clones has recentlybeen described (Altman et al., Science 274:94-96, 1996; Dunbar et al.,Curr. Biol. 8:413-416, 1998), in which fluorogenic tetramers of MHCclass I molecule/peptide complexes are used to detect specific CTL.clones. Briefly, soluble MHC class I molecules are folded in vitro inthe presence of β₂-microglobulin and a peptide antigen which binds theclass I molecule. After purification, the MHC/peptide complex ispurified and labeled with biotin. Tetramers are formed by mixing thebiotinylated peptide-MHC complex with labeled avidin (e.g.phycoerythrin) at a molar ratio or 4:1. Tetramers are then contactedwith a source of CTLs such as peripheral blood or lymph node. Thetetramers bind CTLs which recognize the peptide antigen/MHC class Icomplex. Cells bound by the tetramers can be sorted by fluorescenceactivated cell sorting to isolate the reactive CTLs. The isolated CTLsthen can be expanded in vitro for use as described herein.

To detail a therapeutic methodology, referred to as adoptive transfer(Greenberg, J. Immunol. 136(5): 1917, 1986; Riddel et al., Science 257:238, 1992; Lynch et al, Eur. J. Immunol. 21: 1403-1410,1991; Kast etal., Cell 59: 603-614, 1989), cells presenting the desired complex(e.g., dendritic cells) are combined with CTLs leading to proliferationof the CTLs specific thereto. The proliferated CTLs are thenadministered to a subject with a cellular abnormality which ischaracterized by certain of the abnormal cells presenting the particularcomplex. The CTLs then lyse the abnormal cells, thereby achieving thedesired therapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA/cancer associated antigencomplex. This can be determined very easily, as the art is very familiarwith methods for identifying cells which present a particular HLAmolecule, as well as how to identify cells expressing DNA of thepertinent sequences, in this case a cancer associated antigen sequence.Once cells presenting the relevant complex are identified via theforegoing screening methodology, they can be combined with a sample froma patient, where the sample contains CTLs. If the complex presentingcells are lysed by the mixed CTL sample, then it can be assumed that acancer associated antigen is being presented, and the subject is anappropriate candidate for the therapeutic approaches set forth supra.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo, usinga number of approaches. One approach is the use of non-proliferativecells expressing the complex. The cells used in this approach may bethose that normally express the complex, such as irradiated tumor cellsor cells transfected with one or both of the genes necessary forpresentation of the complex (i.e. the antigenic peptide and thepresenting HLA molecule). Chen et al. (Proc. Natl. Acad. Sci. USA 88:110-114,1991) exemplifies this approach, showing the use of transfectedcells expressing HPVE7 peptides in a therapeutic regime. Various celltypes may be used. Similarly, vectors carrying one or both of the genesof interest may be used. Viral or bacterial vectors are especiallypreferred. For example, nucleic acids which encode a cancer associatedantigen polypeptide or peptide may be operably linked to promoter andenhancer sequences which direct expression of the cancer associatedantigen polypeptide or peptide in certain tissues or cell types. Thenucleic acid may be incorporated into an expression vector. Expressionvectors may be unmodified extrachromosomal nucleic acids, plasmids orviral genomes constructed or modified to enable insertion of exogenousnucleic acids, such as those encoding cancer associated antigen, asdescribed elsewhere herein. Nucleic acids encoding a cancer associatedantigen also may be inserted into a retroviral genome, therebyfacilitating integration of the nucleic acid into the genome of thetarget tissue or cell type. In these systems, the gene of interest iscarried by a microorganism, e.g., a Vaccinia virus, pox virus, herpessimplex virus, retrovirus or adenovirus, and the materials de facto“infect” host cells. The cells which result present the complex ofinterest, and are recognized by autologous CTLs, which then proliferate.

A similar effect can be achieved by combining the cancer associatedantigen or a stimulatory fragment thereof with an adjuvant to facilitateincorporation into antigen presenting cells in vivo. The cancerassociated antigen polypeptide is processed to yield the peptide partnerof the HLA molecule while a cancer associated antigen peptide may bepresented without the need for further processing. Generally, subjectscan receive an intradermal injection of an effective amount of thecancer associated antigen. Initial doses can be followed by boosterdoses, following immunization protocols standard in the art. Preferredcancer associated antigens include those found to react with allogeneiccancer antisera, shown in the examples below.

The invention involves the use of various materials disclosed herein to“immunize” subjects or as “vaccines”. As used herein, “immunization” or“vaccination” means increasing or activating an immune response againstan antigen. It does not require elimination or eradication of acondition but rather contemplates the clinically favorable enhancementof an immune response toward an antigen. Generally accepted animalmodels can be used for testing of immunization against cancer using acancer associated antigen nucleic acid. For example, human cancer cellscan be introduced into a mouse to create a tumor, and one or more cancerassociated antigen nucleic acids can be delivered by the methodsdescribed herein. The effect on the cancer cells (e.g., reduction oftumor size) can be assessed as a measure of the effectiveness of thecancer associated antigen nucleic acid immunization. Of course, testingof the foregoing animal model using more conventional methods forimmunization include the administration of one or more cancer associatedantigen polypeptides or peptides derived therefrom, optionally combinedwith one or more adjuvants and/or cytokines to boost the immuneresponse. Methods for immunization, including formulation of a vaccinecomposition and selection of doses, route of administration and theschedule of administration (e.g. primary and one or more booster doses),are well known in the art. The tests also can be performed in humans,where the end point is to test for the presence of enhanced levels ofcirculating CTLs against cells bearing the antigen, to test for levelsof circulating antibodies against the antigen, to test for the presenceof cells expressing the antigen and so forth.

As part of the immunization compositions, one or more cancer associatedantigens or stimulatory fragments thereof are administered with one ormore adjuvants to induce an immune response or to increase an immuneresponse. An adjuvant is a substance incorporated into or administeredwith antigen which potentiates the immune response. Adjuvants mayenhance the immunological response by providing a reservoir of antigen(extracellularly or within macrophages), activating macrophages andstimulating specific sets of lymphocytes. Adjuvants of many kinds arewell known in the art. Specific examples of adjuvants includemonophosphoryl lipid A (MPL, SmithKline Beecham), a congener obtainedafter purification and acid hydrolysis of Salmonella minnesota Re 595lipopolysaccharide; saponins including QS21 (SmithKline Beecham), a pureQA-21 saponin purified from Quillja saponaria extract; DQS21, describedin PCT application W096/33739 (SmithKline Beecham); QS-7, QS-17, QS-18,and QS-L1 (So et al., Mol. Cells 7:178-186, 1997); incomplete Freund'sadjuvant; complete Freund's adjuvant; montanide; and variouswater-in-oil emulsions prepared from biodegradable oils such as squaleneand/or tocopherol. Preferably, the peptides are administered mixed witha combination of DQS21/MPL. The ratio of DQS21 to MPL typically will beabout 1:10 to 10:1, preferably about 1:5 to 5:1 and more preferablyabout 1:1. Typically for human administration, DQS21 and MPL will bepresent in a vaccine formulation in the range of about 1 μg to about 100μg. Other adjuvants are known in the art and can be used in theinvention (see, e.g. Goding, Monoclonal Antibodies: Principles andPractice, 2nd Ed., 1986). Methods for the preparation of mixtures oremulsions of peptide and adjuvant are well known to those of skill inthe art of vaccination.

Other agents which stimulate the immune response of the subject can alsobe administered to the subject. For example, other cytokines are alsouseful in vaccination protocols as a result of their lymphocyteregulatory properties. Many other cytokines useful for such purposeswill be known to one of ordinary skill in the art, includinginterleukin-12 (IL-12) which has been shown to enhance the protectiveeffects of vaccines (see, e.g., Science 268: 1432-1434, 1995), GM-CSFand IL-18. Thus cytokines can be administered in conjunction withantigens and adjuvants to increase the immune response to the antigens.

There are a number of immune response potentiating compounds that can beused in vaccination protocols. These include costimulatory moleculesprovided in either protein or nucleic acid form. Such costimulatorymolecules include the B7-1 and B7-2 (CD80 and CD86 respectively)molecules which are expressed on dendritic cells (DC) and interact withthe CD28 molecule expressed on the T cell. This interaction providescostimulation (signal 2) to an antigen/MHC/TCR stimulated (signal 1) Tcell, increasing T cell proliferation and effector function. B7 alsointeracts with CTLA4 (CD152) on T cells and studies involving CTLA4 andB7 ligands indicate that the B7-CTLA4 interaction can enhance antitumorimmunity and CTL proliferation (Zheng P., et al. Proc. Natl. Acad. Sci.USA 95 (11):6284-6289 (1998)).

B7 typically is not expressed on tumor cells so they are not efficientantigen presenting cells (APCs) for T cells. Induction of B7 expressionwould enable the tumor cells to stimulate more efficiently CTLproliferation and effector function. A combination of B7/IL-6/IL-12costimulation has been shown to induce IFN-gamma and a Th1 cytokineprofile in the T cell population leading to further enhanced T cellactivity (Gajewski et al., J. Immunol, 154:5637-5648 (1995)). Tumor celltransfection with B7 has ben discussed in relation to in vitro CTLexpansion for adoptive transfer immunotherapy by Wang et al., (J.Immunol., 19:1-8 (1986)). Other delivery mechanisms for the B7 moleculewould include nucleic acid (naked DNA) immunization (Kim J., et al. NatBiotechnol., 15:7:641-646 (1997)) and recombinant viruses such as adenoand pox (Wendtner et al., Gene Ther., 4:7:726-735 (1997)). These systemsare all amenable to the construction and use of expression cassettes forthe coexpression of B7 with other molecules of choice such as theantigens or fragment(s) of antigens discussed herein (includingpolytopes) or cytokines. These delivery systems can be used forinduction of the appropriate molecules in vitro and for in vivovaccination situations. The use of anti-CD28 antibodies to directlystimulate T cells in vitro and in vivo could also be considered.Similarly, the inducible co-stimulatory molecule ICOS which induces Tcell responses to foreign antigen could be modulated, for example, byuse of anti-ICOS antibodies (Hutloff et al., Nature 397:263-266, 1999).

Lymphocyte function associated antigen-3 (LFA-3) is expressed on APCsand some tumor cells and interacts with CD2 expressed on T cells. Thisinteraction induces T cell IL-2 and IFN-gamma production and can thuscomplement but not substitute, the B7/CD28 costimulatory interaction(Parra et al., J. Immunol., 158:637-642 (1997), Fenton et al., J.Immunother., 21:2:95-108 (1998)).

Lymphocyte function associated antigen-1 (LFA-1) is expressed onleukocytes and interacts with ICAM-1 expressed on APCs and some tumorcells. This interaction induces T cell IL-2 and IFN-gamma production andcan thus complement but not substitute, the B7/CD28 costimulatoryinteraction (Fenton et al., J. Immunother., 21:2:95-108 (1998)). LFA-1is thus a further example of a costimulatory molecule that could beprovided in a vaccination protocol in the various ways discussed abovefor B7.

Complete CTL activation and effector function requires Th cell helpthrough the interaction between the Th cell CD40L (CD40 ligand) moleculeand the CD40 molecule expressed by DCs (Ridge et al., Nature, 393:474(1998), Bennett et al., Nature, 393:478 (1998), Schoenberger et al.,Nature, 393:480 (1998)). This mechanism of this costimulatory signal islikely to involve upregulation of B7 and associated IL-6/IL-12production by the DC (APC). The CD40-CD40L interaction thus complementsthe signal 1 (antigen/MHC-TCR) and signal 2 (B7-CD28) interactions.

The use of anti-CD40 antibodies to stimulate DC cells directly, would beexpected to enhance a response to tumor antigens which are normallyencountered outside of a inflammatory context or are presented bynon-professional APCs (tumor cells). In these situations Th help and B7costimulation signals are not provided. This mechanism might be used inthe context of antigen pulsed DC based therapies or in situations whereTh epitopes have not been defined within known TRA precursors.

A cancer associated antigen polypeptide, or a fragment thereof, also canbe used to isolate their native binding partners. Isolation of suchbinding partners may be performed according to well-known methods. Forexample, isolated cancer associated antigen polypeptides can be attachedto a substrate (e.g., chromatographic media, such as polystyrene beads,or a filter), and then a solution suspected of containing the bindingpartner may be applied to the substrate. If a binding partner which caninteract with cancer associated antigen polypeptides is present in thesolution, then it will bind to the substrate-bound cancer associatedantigen polypeptide. The binding partner then may be isolated.

It will also be recognized that the invention embraces the use of thecancer associated antigen cDNA sequences in expression vectors, as wellas to transfect host cells and cell lines, be these prokaryotic (e.g.,E. coli), or eukaryotic (e.g., dendritic cells, B cells, CHO cells, COScells, yeast expression systems and recombinant baculovirus expressionin insect cells). Especially useful are mammalian cells such as human,mouse, hamster, pig, goat, primate, etc. They may be of a wide varietyof tissue types, and include primary cells and cell lines. Specificexamples include keratinocytes, peripheral blood leukocytes, bone marrowstem cells and embryonic stem cells. The expression vectors require thatthe pertinent sequence, i.e., those nucleic acids described supra, beoperably linked to a promoter.

The invention also contemplates delivery of nucleic acids, polypeptidesor peptides for vaccination. Delivery of polypeptides and peptides canbe accomplished according to standard vaccination protocols which arewell known in the art. In another embodiment, the delivery of nucleicacid is accomplished by ex vivo methods, i.e. by removing a cell from asubject, genetically engineering the cell to include a breast cancerassociated antigen, and reintroducing the engineered cell into thesubject. One example of such a procedure is outlined in U.S. Pat. No.5,399,346 and in exhibits submitted in the file history of that patent,all of which are publicly available documents. In general, it involvesintroduction in vitro of a functional copy of a gene into a cell(s) of asubject, and returning the genetically engineered cell(s) to thesubject. The functional copy of the gene is under operable control ofregulatory elements which permit expression of the gene in thegenetically engineered cell(s). Numerous transfection and transductiontechniques as well as appropriate expression vectors are well known tothose of ordinary skill in the art, some of which are described in PCTapplication WO95/00654. In vivo nucleic acid delivery using vectors suchas viruses and targeted liposomes also is contemplated according to theinvention.

In preferred embodiments, a virus vector for delivering a nucleic acidencoding a cancer associated antigen is selected from the groupconsisting of adenoviruses, adeno-associated viruses, poxvirusesincluding vaccinia viruses and attenuated poxviruses, Semliki Forestvirus, Venezuelan equine encephalitis virus, retroviruses, Sindbisvirus, and Ty virus-like particle. Examples of viruses and virus-likeparticles which have been used to deliver exogenous nucleic acidsinclude: replication-defective adenoviruses (e.g., Xiang et al.,Virology 219:220-227, 1996; Eloit et al., J. Virol. 7:5375-5381, 1997;Chengalvala et al., Vaccine 15:335-339; 1997), a modified retrovirus(Townsend et al., J. Virol. 71:3365-3374, 1997), a nonreplicatingretrovirus (Irwin et al., J. Virol. 68:5036-5044, 1994), a replicationdefective Semliki Forest virus (Zhao et al., Proc. Natl. Acad. Sci. USA92:3009-3013, 1995), canarypox virus and highly attenuated vacciniavirus derivative (Paoletti, Proc. Natl. Acad. Sci. USA 93:11349-11353,1996), non-replicative vaccinia virus (Moss, Proc. Natl. Acad. Sci. USA93:11341-11348, 1996), replicative vaccinia virus (Moss, Dev. Biol.Stand. 82:55-63, 1994), Venzuelan equine encephalitis virus (Davis etal., J. Virol. 70:3781-3787, 1996), Sindbis virus (Pugachev et al.,Virology 212:587-594, 1995), and Ty virus-like particle (Allsopp et al.,Eur. J. Immunol 26:1951-1959, 1996). In preferred embodiments, the virusvector is an adenovirus.

Another preferred virus for certain applications is the adeno-associatedvirus, a double-stranded DNA virus. The adeno-associated virus iscapable of infecting a wide range of cell types and species and can beengineered to be replication-deficient. It further has advantages, suchas heat and lipid solvent stability, high transduction frequencies incells of diverse lineages, including hematopoietic cells, and lack ofsuperinfection inhibition thus allowing multiple series oftransductions. The adeno-associated virus can integrate into humancellular DNA in a site-specific manner, thereby minimizing thepossibility of insertional mutagenesis and variability of inserted geneexpression. In addition, wild-type adeno-associated virus infectionshave been followed in tissue culture for greater than 100 passages inthe absence of selective pressure, implying that the adeno-associatedvirus genomic integration is a relatively stable event. Theadeno-associated virus can also function in an extrachromosomal fashion.

In general, other preferred viral vectors are based on non-cytopathiceukaryotic viruses in which non-essential genes have been replaced withthe gene of interest. Non-cytopathic viruses include retroviruses, thelife cycle of which involves reverse transcription of genomic viral RNAinto DNA with subsequent proviral integration into host cellular DNA.Adenoviruses and retroviruses have been approved for human gene therapytrials. In general, the retroviruses are replication-deficient (i.e.,capable of directing synthesis of the desired proteins, but incapable ofmanufacturing an infectious particle). Such genetically alteredretroviral expression vectors have general utility for thehigh-efficiency transduction of genes in vivo. Standard protocols forproducing replication-deficient retroviruses (including the steps ofincorporation of exogenous genetic material into a plasmid, transfectionof a packaging cell lined with plasmid, production of recombinantretroviruses by the packaging cell line, collection of viral particlesfrom tissue culture media, and infection of the target cells with viralparticles) are provided in Kriegler, M., “Gene Transfer and Expression,A Laboratory Manual,” W. H. Freeman Co., New York (1990) and Murry, E.J. Ed. “Methods in Molecular Biology,” vol. 7, Humana Press, Inc.,Cliffton, New Jersey (1991).

Preferably the foregoing nucleic acid delivery vectors: (1) containexogenous genetic material that can be transcribed and translated in amammalian cell and that can induce an immune response in a host, and (2)contain on a surface a ligand that selectively binds to a receptor onthe surface of a target cell, such as a mammalian cell, and therebygains entry to the target cell.

Various techniques may be employed for introducing nucleic acids of theinvention into cells, depending on whether the nucleic acids areintroduced in vitro or in vivo in a host. Such techniques includetransfection of nucleic acid-CaPO₄ precipitates, transfection of nucleicacids associated with DEAE, transfection or infection with the foregoingviruses including the nucleic acid of interest, liposome mediatedtransfection, and the like. For certain uses, it is preferred to targetthe nucleic acid to particular cells. In such instances, a vehicle usedfor delivering a nucleic acid of the invention into a cell (e.g., aretrovirus, or other virus; a liposome) can have a targeting moleculeattached thereto. For example, a molecule such as an antibody specificfor a surface membrane protein on the target cell or a ligand for areceptor on the target cell can be bound to or incorporated within thenucleic acid delivery vehicle. Preferred antibodies include antibodieswhich selectively bind a cancer associated antigen, alone or as acomplex with a MIIC molecule. Especially preferred are monoclonalantibodies. Where liposomes are employed to deliver the nucleic acids ofthe invention, proteins which bind to a surface membrane proteinassociated with endocytosis may be incorporated into the liposomeformulation for targeting and/or to facilitate uptake. Such proteinsinclude capsid proteins or fragments thereof tropic for a particularcell type, antibodies for proteins which undergo internalization incycling, proteins that target intracellular localization and enhanceintracellular half life, and the like. Polymeric delivery systems alsohave been used successfuilly to deliver nucleic acids into cells, as isknown by those skilled in the art. Such systems even permit oraldelivery of nucleic acids.

When administered, the therapeutic compositions of the present inventioncan be administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. When antibodies are used therapeutically, a preferred routeof administration is by pulmonary aerosol. Techniques for preparingaerosol delivery systems containing antibodies are well known to thoseof skill in the art. Generally, such systems should utilize componentswhich will not significantly impair the biological properties of theantibodies, such as the paratope binding capacity (see, for example,Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences,18th edition, 1990, pp 1694-1712; incorporated by reference). Those ofskill in the art can readily determine the various parameters andconditions for producing antibody aerosols without resort to undueexperimentation. When using antisense preparations of the invention,slow intravenous administration is preferred.

The compositions of the invention are administered in effective amounts.An “effective amount” is that amount of a cancer associated antigencomposition that alone, or together with further doses, produces thedesired response, e.g. increases an immune response to the cancerassociated antigen. In the case of treating a particular disease orcondition characterized by expression of one or more cancer associatedantigens, such as small cell lung cancer, the desired response isinhibiting the progression of the disease. This may involve only slowingthe progression of the disease temporarily, although more preferably, itinvolves halting the progression of the disease permanently. This can bemonitored by routine methods or can be monitored according to diagnosticmethods of the invention discussed herein. The desired response totreatment of the disease or condition also can be delaying the onset oreven preventing the onset of the disease or condition.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practioner. These factors are wellknown to those of ordinary skill in the art and can be addressed with nomore than routine experimentation. It is generally preferred that amaximum dose of the individual components or combinations thereof beused, that is, the highest safe dose according to sound medicaljudgment. It will be understood by those of ordinary skill in the art,however, that a patient may insist upon a lower dose or tolerable dosefor medical reasons, psychological reasons or for virtually any otherreasons.

The pharmaceutical compositions used in the foregoing methods preferablyare sterile and contain an effective amount of cancer associated antigenor nucleic acid encoding cancer associated antigen for producing thedesired response in a unit of weight or volume suitable foradministration to a patient. The response can, for example, be measuredby determining the immune response following administration of thecancer associated antigen composition via a reporter system by measuringdownstream effects such as gene expression, or by measuring thephysiological effects of the cancer associated antigen composition, suchas regression of a tumor or decrease of disease symptoms. Other assayswill be known to one of ordinary skill in the art and can be employedfor measuring the level of the response.

The doses of cancer associated antigen compositions (e.g., polypeptide,peptide, antibody, cell or nucleic acid) administered to a subject canbe chosen in accordance with different parameters, in particular inaccordance with the mode of administration used and the state of thesubject. Other factors include the desired period of treatment. In theevent that a response in a subject is insufficient at the initial dosesapplied, higher doses (or effectively higher doses by a different, morelocalized delivery route) may be employed to the extent that patienttolerance permits.

In general, for treatments for eliciting or increasing an immuneresponse, doses of cancer associated antigen are formulated andadministered in doses between 1 ng and 1 mg, and preferably between 10ng and 100 μg, according to any standard procedure in the art. Wherenucleic acids encoding cancer associated antigen of variants thereof areemployed, doses of between 1 ng and 0.1 mg generally will be formulatedand administered according to standard procedures. Other protocols forthe administration of cancer associated antigen compositions will beknown to one of ordinary skill in the art, in which the dose amount,schedule of injections, sites of injections, mode of administration(e.g., intra-tumoral) and the like vary from the foregoing.Administration of cancer associated antigen compositions to mammalsother than humans, e.g. for testing purposes or veterinary therapeuticpurposes, is carried out under substantially the same conditions asdescribed above.

Where cancer associated antigen peptides are used for vaccination, modesof administration which effectively deliver the cancer associatedantigen and adjuvant, such that an immune response to the antigen isincreased, can be used. For administration of a cancer associatedantigen peptide in adjuvant, preferred methods include intradermal,intravenous, intramuscular and subcutaneous administration. Althoughthese are preferred embodiments, the invention is not limited by theparticular modes of administration disclosed herein. Standard referencesin the art (e.g., Remington 's Pharmaceutical Sciences, 18th edition,1990) provide modes of administration and formulations for delivery ofimmunogens with adjuvant or in a non-adjuvant carrier.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptable compositions. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients. Suchpreparations may routinely contain salts, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

A small cell lung cancer associated antigen composition may be combined,if desired, with a pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation of breast cancerassociated antigen polypeptides or nucleic acids, which is preferablyisotonic with the blood of the recipient. This preparation may beformulated according to known methods using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationalso may be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butane diol. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono-or di-glycerides. In addition,fatty acids such as oleic acid may be used in the preparation ofinjectables. Carrier formulation suitable for oral, subcutaneous,intravenous, intramuscular, etc. administrations can be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

As used herein with respect to nucleic acids, the term “isolated” means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art. An isolated nucleic acid as used herein is not a naturallyoccurring chromosome.

As used herein with respect to polypeptides, “isolated” means separatedfrom its native environment and present in sufficient quantity to permitits identification or use. Isolated, when referring to a protein orpolypeptide, means, for example: (i) selectively produced by expressioncloning or (ii) purified as by chromatography or electrophoresis.Isolated proteins or polypeptides may, but need not be, substantiallypure. The term “substantially pure” means that the proteins orpolypeptides are essentially free of other substances with which theymay be found in nature or in vivo systems to an extent practical andappropriate for their intended use. Substantially pure polypeptides maybe produced by techniques well known in the art. Because an isolatedprotein may be admixed with a pharmaceutically acceptable carrier in apharmaceutical preparation, the protein may comprise only a smallpercentage by weight of the preparation. The protein is nonethelessisolated in that it has been separated from the substances with which itmay be associated in living systems, i.e. isolated from other proteins.

EXAMPLES

Methods and Materials

Cell Lines, Tissues, and Patient Sera

Cell lines were obtained from the repository maintained at the LudwigInstitute for Cancer Research (LICR), New York Branch at the MemorialSloan-Kettering Cancer Center (MSKCC), or obtained from American TissueCulture Collection. Eleven SCLC cell lines were used including 9classical (SK-LC-13, NCI-H69, -H128, -H146, -H187, -H209, -H378, -H889,-H740) and 2 variant (NCI-H82, -H526) forms. The variant SCLC linesdiffer from the classical lines in lacking or having diminishedneuroendocrine features and with regard to other biochemical,morphological and growth properties (Carney et al., Cancer Res.45:2913-2923, 1985; Park et al., Cancer Res. 47:6710-6718, 1987). Normaland tumor tissues were obtained from the departments of Pathology in theNew York Presbyterian Hospital (NYPH) and the MSKCC. Patient sera wereobtained from the Department of Medicine, NYPH, and from the LICRMelbourne Branch, Australia.

Immunoscreening of the SCLC Cell Line Libraries and Characterization ofImmunoreactive Clones

Construction of cDNA expression libraries from the NCI-H740 and SK-LC-13SCLC cell lines in the λ-ZAP vector (Stratagene) and immunoscreeningwere done as previously described (Old and Chen, J. Exp. Med.,187:1163-7, 1998; Chen et al., Proc. Nat'l. Acad. Sci. USA, 95: 6919-23,1998), with the following modifications. Sera from five SCLC patients(Lu94, Lu100, Lu101, Lu104, Lu113) were pooled and absorbed aspreviously described Scanlan et al., Int. J. Cancer 76:652-658, 1998).The pooled serum was diluted 1:200 (final dilution 1:1000 for eachserum) in TBS containing 1% BSA and 0.02% NaN₃ and was used to screen5.6×10⁵ pfu of the NCI-H740 library. The same serum was used for theSK-LC-13 library of which 2.2×10⁵ pfu was screened. Immunoreactiveclones were isolated and sequence analyzed as previously described (Chenet al., 1998). Selected immunoreactive clones were subsequently testedfor reactivity against sera at various dilutions from individual lungcancer patients and normals using the same plaque assay. A λ-ZAP clonewithout an insert was co-plated and included in the screen as a negativecontrol.

RT-PCR Analysis

Reverse transcription was performed with total RNA isolated from tissueor cell lines by the Guanidium thiocyanate/CsCl method. Primers used toamplify ZIC2 were designed based on the published sequence (AF104902)and our results. ZIC2A1:5′-CATGAATATGAACATGGGTATGAACATGG (SEQ ID NO:1);ZIC2B1:5′-TCGCAGCCCTCAAACTCACACTG (SEQ ID NO:2). Conditions foramplification were as follows: Initial denaturation and AmpliTaq Gold(Perkin Elmer) activation; 94° C., 10′, Amplification: 94° C., 1′; 60°C., 1′; 72° C., 1′; for 35 cycles, followed by a 6′, 72° C. incubation.Amplification products were analyzed by agarose gel electrophoresis andvisualized by EtBr staining.

Northern Blot Analysis

Adult normal tissue MRNA blots were obtained from Clontech, Inc. andcontained 2 g polyA⁺ RNA per lane. Lung cancer cell line total RNA wasisolated as described above and polyA⁺ mRNA was prepared using theMicrofast Track kit (Invitrogen). Two grams of mRNA or 10 g of total RNAwas transferred to nylon membranes (Schleicher and Schuell) followingdenaturing gel electrophoresis. Hybridizations and washes were carriedout under high stringency conditions in ExpressHyb buffer (Clontech)using hybridization and washing conditions described by themanufacturer. The probes used for northern blot analysis were thefollowing. SOX2: 450 bp fragment (nucleotides 630-1080); SOX1: 751 bpfragment (nucleotides 1520-2271); SOX3: 330 bp fragment (nucleotides442-772); SOX21: 680 bp fragment (nucleotides 2720-3400); and ID4:full-length cDNA (1322 bp).

Example 1 Isolation of Immunoreactive Clones from SCLC Cell Lines bySEREX

SEREX analysis of the SCLC cell line NCI-H740 with a pool of five serafrom SCLC patients at 1:10³ dilution resulted in the isolation of 37clones coding for 8 known gene products (Table 1a). These eight geneswere given SEREX gene designations of NIVY-SCLC-1 to NY-SCLC-8.

TABLE 1a Genes isolated by SEREX analysis of the small cell lung cancercell line NCI-H740 SEQ ID Gene Gene/Sequence Identity Number of clonesNO: Designation [GenBank Accession No.] (% of total) 3 NY-SCLC-1 SOX2[Z31560] 19 (51%) 4 NY-SCLC-2 SOX1 [Y13436]  1 (3%) 5 NY-SCLC-3 ZIC2[AF104902]  9 (24%) 6 NY-SCLC-4 ID4 [U28368]  2 (5%) 7 NY-SCLC-5 MAZ[M94046]  1 (3%) 8 NY-SCLC-6 MPP11 [X98260]  3 (8%) 9 NY-SCLC-7 eIF2B[U23028]  1 (3%) 10 NY-SCLC-8 RBP-1 [L07872]  1 (3%) Total: 37

The most frequently isolated genes were SOX2 and ZIC2, comprising 51%and 24% of all clones. A single clone corresponding to SOX1 was alsoisolated from this library. SOX- and ZIC2-encoding clones showed verystrong immunoreactivity with the SCLC patient sera. Other genes isolatedincluded ID4, MPP11, MAZ, eIF2B and RBP-1. ID4 protein is a member ofthe dominant negative helix-loop-helix (HLH) proteins. This protein caninteract with other HLH proteins such as the one encoded byArchaete-Scute and by virtue of not containing a DNA binding domain itacts as a repressor (Riechmann, et al., Nucleic Acids Res., 22: 749-55,1994). The mRNA expression pattern of ID4 in normal tissues was found tobe universal by Northern blot analysis. Seroreactivity against ID4 wasmoderate at 1:10³ sera dilution. MPP11 is another HLH protein-bindingfactor, and it has also been isolated from HeLa cells by M-phaseprotein-recognizing antibodies (Shoji, et al., J. Biol. Chem.,270:24818-25, 1995; Matsumoto-Taniura, et al. Mol. Biol. Cell, 7:1455-69, 1996). Seroreactivity against MPP11 was strong at a 1:1000dilution of the SCLC sera. This antigen was also identified by SEREXanalysis of gastric and breast cancer and is universally expressed.Other genes isolated from NCI-H740—the myc-associated Zinc-fingerprotein MAZ, the eukaryotic translation initiation factor eIF2B and theJ-κ recombination signal binding protein (RBP-1)—were also previouslyidentified by SEREX. MAZ, eIF2B and RBP-1 are expressed in multiplenormal adult tissues.

The SEREX analysis of the second SCLC line SK-LC-13 with the same pooledsera from SCLC patients resulted in the identification of 14 clonescorresponding to 10 genes (Table Ib), 4 of which were identical to thoseisolated from NCI-H740 and 6 were distinct (NY-SCLC-9 to NY-SCLC-14).

TABLE 1b Genes isolated by SEREX analysis of the small cell lung cancercell line SK-LC-13 SEQ ID Gene Gene/Sequence Identity Number of clonesNO: Designation [GenBank Accession No.] (% of total)  3 NY-SCLC-1 SOX2[Z31560]  2 (14%) 11 NY-SCLC-9 SOX3 [X71135]  1 (7%) 12 NY-SCLC-10 SOX21[AF107044]  1 (7%)  5 NY-SCLC-3 ZIC2 [AF104902]  2 (14%)  6 NY-SCLC-4ID4 [U28368]  1 (7%)  8 NY-SCLC-6 MPP11 [X98260]  3 (21%) 13 NY-SCLC-11KIAA0963 [AB023180.1]  1 (7%) 14 NY-SCLC-12 LAG-3 [X51985]  1 (7%) 15,16 NY-SCLC-13 DKFZp434C196  1 (7%) [AL133561.1] 17 NY-SCLC-14 Novel-2  1(7%) Total: 14

SOX2 was isolated twice and in addition SOX3 and SOX21 were isolated,each represented by a single clone. ZIC2 was isolated twice. Other genesisolated that were identical to those from the NCI-H740 library includedID4, isolated once, and MPP11, which was represented by threeimmunoreactive clones. Among other genes identified, NY-SCLC-11(KIAA0963) is an unknown gene with identical EST sequences derived frommany tissues. Two novel genes (NY-SCLC-13) and (NY-SCLC-14) wereisolated, one of which (NY-SCLC-14) showed no sequence identity tocurrent GenBank entries. These two genes were intriguing in that theirDNA sequences contain homopolymers of 24 bp and 6 bp repeats and wouldencode tandem octapeptides and dipeptides, respectively. NY-SCLC-12,lymphocyte activation gene-3 (LA G-3), is related to CD4 and has arestricted tissue expression pattern, possibly representing adifferentiation antigen of lymphoid origin (Triebel, et al., J. Exp.Med., 171: 1393-1405, 1990).

Example 2 Immunodominant Epitopes of ZIC2 and the SOX Proteins

Of 11 ZIC2 clones isolated, 7 clones were sequenced and 4 were evaluatedby restriction mapping. The longest ZIC2 clone (NCI-H740 #32) was ˜2.6kb, the sequence of which extends beyond both 5′ and 3′ sequences of theZIC2 cDNA entry in the GenBank (AF104902). The shortest clone (NCI-H740#41) migrated as a ˜1 kb band on agarose gels and its 5′ endcorresponded to nucleotide position 692 (amino acid residue 231) ofAF104902. Reactivity of this clone with SCLC sera was comparable toother larger clones. As the intensity of the reactivity of this shorterclone was comparable to that of other larger ZIC2 clones, theseroreactive epitope(s) of ZIC2 polypeptide (SEQ ID NO:22) residebetween amino acid residue 231 and the C-terminal end (amino acidresidue 533).

Of the 24 SOX genes, 8 SOX2 clones and the SOX1, SOX3 and SOX21 cloneswere sequence analyzed while the remaining 13 SOX2 clones were analyzedand confirmed by restriction mapping. All SOX2 clones contained the fullsize cDNA (1085 bp) and the longest clone (NCI-H740 #2) had 54additional nucleotides at its 5′ untranslated region as compared to theSOX2 GenBank entry (Accession Number Z31560). The two SOX1 and SOX3clones contained truncated cDNA inserts which lacked sequences 5′ tothose encoding the HMG-box while the SOX21 clone encoded the full lengthSOX21 protein, which has only 5 residues N-terminal to its HMG-box (FIG.1). The most conserved region among these SOX cDNA clones is thus theHMG-box-encoding region which is 88 to 96% identical among the SOX GroupB proteins. All sera that reacted with SOX1 also reacted with SOX2, SOX3and SOX21 (see below), suggesting that at least part of theimmunoreactivity of SCLC patient sera is directed against the conservedHMG-box of the SOX proteins.

Example 3 ZIC2 is Expressed Exclusively in Brain, Testis and Tumors

ZIC2 gene expression was analyzed by RT-PCR. The RNA quality wasconfirmed by successful amplification of p53 exons 5 and 6. Among normaltissues ZIC2 mRNA was only detectable in brain and to a lesser extent intestis but not in skin, kidney, small intestine, pancreas, uterus andlung. Of 11 SCLC cell lines analyzed, all 9 classical SCLC lines(SK-LC-13, NCI-H69, -H128, -H146, -H187, -H209, -H378, -H889, -H740) haddetectable ZIC2 mRNA while two variant SCLC cell lines (NCI-H82 andNCI-H526) showed no or minimal expression. Among other cell lines, ZIC2mRNA could be amplified in 100% ( 7/7) of non-small cell lung tumor celllines and 83% ( 10/12) of melanoma cell lines (Table 2). Among tumortissues, 50% ( 5/10) of melanoma, 50% ( 2/4) of colon cancer, 75% (¾) ofbreast cancer, 86% ( 12/14) of head and neck cancer, 66% ( 6/9) of lungcancer, 50% ( 7/14) of transitional cancer, 50% (½) of leiomyosarcomaand 100% ( 2/2) of synovial sarcoma samples had detectable ZIC2 mRNA(Table 2).

TABLE 2 ZIC2 gene expression in cancer cell lines and tumor samples ZIC2mRNA EXPRESSION TUMOR CELL LINE Melanoma 10/12 (83%) NSCLC  7/7 (100%)TUMOR TYPE Melanoma  5/10 (50%) Colon cancer  2/4 (50%) Breast cancer 3/4 (75%) Head & neck cancer 12/14 (86%) Lung cancer  6/9 (66%)Transitional cancer  7/14 (50%) Leiomyosarcoma  1/2 (50%) Synovialsarcoma  2/2 (100%)

Example 4 SOX Gene Expression Characteristics

Since SOX Group B genes are intronless, RT-PCR results using tissue RNAwere often difficult to interpret due to the genomic DNA contaminationof RNA samples. Therefore, their gene expression was evaluated byNorthern blot analysis. An α-actin probe was used to confirm the RNAquality and quantity. Northern blots were exposed for 24 h (SOX2-SCLCblot), 72 h (SOX1), or 1 week (SOX3, SOX21 and SOX2-normal tissue blot).

Among normal tissues SOX2 mRNA could be detected in brain, testis andprostate, and at lower levels in small intestine and colon but not inheart, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,thymus, ovary and peripheral blood leukocytes. SOX1, SOX3 and SOX21 mRNAwere not detected in normal adult tissues, which is consistent with thecurrent literature. SOX Group B expression in tumor cell lines was alsoexamined. SOX2 was expressed in 5 of 10 SCLC cell lines (NCI-H69,NCI-H146, NCI-H378, NCI-H740 and SK-LC-13). SOX2 message was notdetected in the three non-SCLC cell lines SK-LC-7, 8 and 17 or in the 8melanoma cell lines SK-MEL-10, 12, 14, 24, 26, 28, 37 and Mz19. SOX1mRNA was detected in 4 of 10 SCLC cell lines (NCI-H187, NCI-H209,NCI-H378 and SK-LC-13) while SOX3 mRNA could be detected in 2 of 10 SCLCcell lines (NCI-H740, and as a weaker signal in SK-LC-13). SOX1 and SOX3required longer exposure times than SOX2, indicating their expressionlevels are lower than that of SOX2. SOX21 mRNA was not detected afterprolonged exposure (1 week), indicating no or low levels of expression.Two variant SCLC cell lines, NCI-H82 and NCI-H526, had no detectable SOXGroup B expression.

Example 5 SCLC Patient Sera Contain High-titer Antibodies to SOX andZIC2 Proteins

Reactivity to phage clones containing SOX1, 2, 3, 21 and ZIC2 wastitered against 17 SCLC patient sera and 16 normal adult sera. ZAPphages with no insert were mixed with the test clone and served asinternal negative controls, visible as a background at 1:10⁴serodilution on Lu113. Assays were scored positive only when test clonescould be clearly distinguished from the control phages.

Only one of the 16 normal sera showed weak reactivity against SOX2 at atiter of 1:1000. In contrast, 7 of 17 patients (41%) had antibodiesreactive with SOX1 and SOX2 containing phagemids while 29% ( 5/17) and35% ( 6/17) had antibodies to SOX3 and SOX21 respectively. 29% ( 5/17)of patients had detectable anti-ZIC2 antibodies. The antibody titersmeasured up to 1:106 (Table 3). All five patient sera that hadantibodies against ZIC2 also reacted with SOX proteins at varyingtiters; one (Lu113) was reactive at 1:106 while another (Lu139) wasreactive only at a 1:10³ dilution. Two patients (Lu100 and A6) hadantibodies against SOX1 and SOX2 proteins at 1:105 but no antibodiesagainst ZIC2 even at 1:10³ dilution (Table 3).

TABLE 3 SOX and ZIC2 Reactivity of Small Cell Lung Cancer Patient SeraProtein: Serum: SOX1 SOX2 SOX3 SOX21 ZIC2 1 Lu 94* 1:10⁵ 1:10⁵ 1:10⁴1:10⁴ 1:10⁵ 2 Lu 100* 1:10⁵ 1:10⁵ 1:10⁴ 1:10⁴ — 3 Lu 101* — — — — — 4 Lu104* — — — — — 5 Lu 113* 1:10⁶ 1:10⁶ 1:10⁵ 1:10⁵ 1:10⁶ 6 Lu 139 1:10³1:10³ — — 1:10⁶ 7 Lu 159 — — — — — 8  A1 1:10⁵ 1:10⁶ 1:10⁵ 1:10⁵ 1:10⁴ 9 A2 — — — — — 10  A3 — — — — — 11  A4 — — — — — 12  A5 — — — — — 13  A61:10⁵ 1:10⁵ 1:10³ 1:10⁴ — 14  A7 — — — — — 15  A8 — — — — — 16  A9 — — —— — 17 A10 1:10⁴ 1:10⁴ — 1:10³ 1:10⁴ 7/17(41%) 7/17(41%) 5/17(29%)6/17(35%) 5/17(29%) *Pooled sera used for SEREX analysis of the SCLCcell lines

All patients who had antibodies against SOX3 or SOX21 had antibodies athigher titers against SOX1 and SOX2. The presence of consistently highertiter antibodies against SOX1 and SOX2 suggests SOX1 and/or 2 as themain immunogenic tumor antigen in these patients, whereas theseroreactivity to SOX3 and SOX21 might be secondary to the shareantigenic epitopes located within the highly conserved HMG-box among SOXproteins.

From the immunological standpoint, the high frequency and high titers ofanti-SOX and anti-ZIC2 antibodies in these SCLC patients is striking.Anti-ZIC2 antibody was observed in 29% and anti-SOX antibody wasobserved in 41% of the SCLC sera tested. These sera were collected froma heterogeneous group of SCLC patients who were at different stages oftheir diseases, receiving various treatments, and with variableresponses; one of the antibody-positive patients (Lu113) had no clinicalevidence of residual disease when serum was collected and had subsequentrecurrence of tumor. This means that if serum is collected fromuntreated cases of SCLC, the frequency of detecting anti-SOX andanti-ZIC2 antibodies can be substantially higher than the 30-40% ratefound in this study. This frequency is significantly higher than theantibody responses seen against most other SEREX-defined antigens.Scanlan et al. (Int. J. Cancer 76:652-658, 1998) have evaluated largepanels of SEREX antigens for seroreactivity in cancer and normalpatients. It was found that antigens that elicit cancer-specificantibody responses tend to have detectable seral antibody in up to20-25% of tumor patients, rarely exceeding 25%. In this regard, theimmunogenicity of SOX and ZIC2 antigens in these patients areexceptional and this indicates that an antibody-based assay can beuseful in the diagnosis of SCLC, e.g. as a screening test for thehigh-risk group. Also, for SCLC cases that have been shown to havehigh-titer antibodies, the titer of the antibody can be correlated tothe clinical progression/remission of the disease. If the presence ofantibody is dependent on the tumor load, as has been shown for anotherSEREX-defined antigen, NY-ESO-1 (Stockert et al., J. Exp. Med.187:1349-1354, 1998), antibody monitoring in these patients may also beof clinical value.

In addition to its immunodiagnostic potential, SOX group B and ZIC2products can be used as targets for cancer vaccines. The expression ofthese genes in brain may be a concern, particularly given theclinically-recognized paraneoplastic syndromes and their correlation tothe aberrant expression of neural antigens in SCLC (Dalmau & Posner,Arch. Neurol. 56:405-408, 1999; Posner & Dalmau, Curr. Opin. Immunol.9:723-729, 1997). However, despite the presence of high-titer anti-SOXand anti-ZIC2 antibodies, none of the seven antibody-positive patientsin this study had neurological manifestations of the disease. In fact,the only patient in this study with paraneoplastic disease involving thecerebellum (patient A9) had no detectable anti-SOX Group B or anti-ZIC2antibodies. The immune responses toward these antigens thus may not leadto autoimmune neurological disorders in most patients. Since SOX andZIC2 genes are conserved in mice, preclinical studies can be carried outby SOX and/or ZIC2 vaccination in these experimental models. Indeed, HuDantigen, one of the antigens associated with paraneoplastic syndromes,has recently been used as a vaccine target in the murine model of smallcell lung cancer, and antitumor activity was observed withoutneurological disease (Carpentier et al., Clin. Cancer Res. 4:2819-2824,1998; Ohwada et al., Am. J. Respir. Cell. Mol. Biol. 21:37-43,1999).

Example 6 Preparation of Recombinant Cancer Associated Antigens

To facilitate screening of patients' sera for antibodies reactive withcancer associated antigens, for example by ELISA, recombinant proteinsare prepared according to standard procedures. In one method, the clonesencoding cancer associated antigens are subcloned into a baculovirusexpression vector, and the recombinant expression vectors are introducedinto appropriate insect cells. Baculovirus/insect cloning systems arepreferred because post-translational modifications are carried out inthe insect cells. Another preferred eukaryotic system is the DrosophilaExpression System from Invitrogen. Clones which express high amounts ofthe recombinant protein are selected and used to produce the recombinantproteins. The recombinant proteins are tested for antibody recognitionusing serum from the patient which was used to isolated the particularclone, or in the case of cancer associated antigens recognized byallogeneic sera, by the sera from any of the patients used to isolatethe clones or sera which recognize the clones' gene products.

Alternatively, the cancer associated antigen clones are inserted into aprokaryotic expression vector for production of recombinant proteins inbacteria. Other systems, including yeast expression systems andmammalian cell culture systems also can be used.

Example 7 Preparation of Antibodies to Cancer Associated Antigens

The recombinant cancer associated antigens produced as in Example 6above are used to generate polyclonal antisera and monoclonal antibodiesaccording to standard procedures. The antisera and antibodies soproduced are tested for correct recognition of the cancer associatedantigens by using the antisera/antibodies in assays of cell extracts ofpatients known to express the particular cancer associated antigen (e.g.an ELISA assay). These antibodies can be used for experimental purposes(e.g. localization of the cancer associated antigens,immunoprecipitations, Western blots, etc.) as well as diagnosticpurposes (e.g., testing extracts of tissue biopsies, testing for thepresence of cancer associated antigens).

The antibodies are useful for accurate and simple typing of small celllung cancer tissue samples for expression of SOX Group B and ZIC2 genes.SCLC is usually diagnosed by endoscopic biopsies rather than surgicalresection, and an adequate specimen for RNA extraction and RT-PCR typingmay not be obtained in every case. These difficulties are furthercomplicated by the fact that SOX Group B genes are intronless, andRT-PCR is often unreliable. The best technique to type the expression ofthese genes and circumvent these problems is by immunohistochemicalanalysis with specific antibody reagents.

Example 8 Expression of Cancer Associated Antigens in Cancers of Similarand Different Origin

The expression of one or more of the cancer associated antigens istested in a range of tumor samples to determine which, if any, othermalignancies should be diagnosed and/or treated by the methods describedherein. Tumor cell lines and tumor samples are tested for cancerassociated antigen expression, preferably by RT-PCR according tostandard procedures, e.g., as described for ZIC2 expression in Example 3above. Northern blots also are used to test the expression of the cancerassociated antigens. Antibody based assays, such as ELISA and westernblot, also can be used to determine protein expression. A preferredmethod of testing expression of cancer associated antigens (in othercancers and in additional same type cancer patients) is allogeneicserotyping using a modified SEREX protocol (as described above).

In all of the foregoing, extracts from the tumors of patients whoprovided sera for the initial isolation of the cancer associatedantigens are used as positive controls. The cells containing recombinantexpression vectors described in the Examples above also can be used aspositive controls.

The results generated from the foregoing experiments provide panels ofmultiple cancer associated nucleic acids and/or polypeptides for use indiagnostic (e.g. determining the existence of cancer, determining theprognosis of a patient undergoing therapy, etc.) and therapeutic methods(e.g., vaccine composition, etc.).

Example 9 HLA Typing of Patients Positive for Cancer Associated Antigens

To determine which HLA molecules present peptides derived from thecancer associated antigens of the invention, cells of the patients whichexpress the cancer associated antigens are HLA typed. Peripheral bloodlymphocytes are taken from the patient and typed for HLA class I orclass II, as well as for the particular subtype of class I or class II.Tumor biopsy samples also can be used for typing. HLA typing can becarried out by any of the standard methods in the art of clinicalimmunology, such as by recognition by specific monoclonal antibodies, orby HLA allele-specific PCR (e.g. as described in WO97/31126).

Example 10 Characterization of Cancer Associated Antigen PeptidesPresented by MHC Class I and Class II Molecules

Antigens which provoke an antibody response in a subject may alsoprovoke a cell-mediated immune response. Cells process proteins intopeptides for presentation on MHC class I or class II molecules on thecell surface for immune surveillance. Peptides presented by certainMHC/HLA molecules generally conform to motifs. These motifs are known insome cases, and can be used to screen the small cell lung cancerassociated antigens for the presence of potential class I and/or classII peptides. Summaries of class I and class II motifs have beenpublished (e.g., Rammensee et al., Immunogenetics 41:178-228, 1995).Based on the results of experiments such as those described above, theHLA types which present the individual breast cancer associated antigensare known. Motifs of peptides presented by these HLA molecules thus arepreferentially searched.

One also can search for class I and class II motifs using computeralgorithms. For example, computer programs for predicting potential CTLepitopes based on known class I motifs has been described (see, e.g.,Parker at al, J. Immunol. 152:163, 1994; D'Amaro et al., Human Immunol.43:13-18, 1995; Drijfhout et al., Human Immunol. 43:1-12, 1995).Computer programs for predicting potential T cell epitopes based onknown class II motifs has also been described (see, e.g. Sturniolo etal., Nat Biotechnol 17(6):555-61, 1999). HLA binding predictions canconveniently be made using an algorithm available via the Internet onthe National Institutes of Health World Wide Web site. See also thewebsite of: SYFPEITHI: An Jnternet Database for MHC Ligands and PeptideMotifs. Methods for determining HLA class II peptides and makingsubstitutions thereto are also known (e.g. Strominger and Wucherpfennig(PCT/US96/03182)).

Example 11 Identification of the Portion of a Cancer AssociatedPolypeptide Encoding an Antigen

To determine if the cancer associated antigens isolated as describedabove can provoke a cytolytic T lymphocyte response, the followingmethod is performed. CTL clones are generated by stimulating theperipheral blood lymphocytes (PBLs) of a patient with autologous normalcells transfected with one of the clones encoding a cancer associatedantigen polypeptide or with irradiated PBLs loaded with syntheticpeptides corresponding to the putative protein and matching theconsensus for the appropriate HLA class I molecule (as described above)to localize an antigenic peptide within the cancer associated antigenclone (see, e.g., Knuth et al., Proc. Natl. Acad. Sci. USA 81:3511-3515,1984; van derBruggen et al., Eur. J. Immunol. 24:3038-3043, 1994). TheseCTL clones are screened for specificity against COS cells transfectedwith the cancer associated antigen clone and autologous HLA alleles asdescribed by Brichard et al. (Eur. J. Immunol. 26:224-230, 1996). CTLrecognition of a cancer associated antigen is determined by measuringrelease of TNF from the cytolytic T lymphocyte or by ⁵¹Cr release assay(Herin et al., Int. J. Cancer 39:390-396, 1987). If a CTL clonespecifically recognizes a transfected COS cell, then shorter fragmentsof the cancer associated antigen clone transfected in that COS cell aretested to identify the region of the gene that encodes the peptide.Fragments of the cancer associated antigen clone are prepared byexonuclease III digestion or other standard molecular biology methods.Synthetic peptides are prepared to confirm the exact sequence of theantigen.

Optionally, shorter fragments of cancer associated antigen cDNAs aregenerated by PCR. Shorter fragments are used to provoke TNF release or⁵¹Cr release as above.

Synthetic peptides corresponding to portions of the shortest fragment ofthe cancer associated antigen clone which provokes TNF release areprepared. Progressively shorter peptides are synthesized to determinethe optimal cancer associated antigen tumor rejection antigen peptidesfor a given HLA molecule.

A similar method is performed to determine if the cancer associatedantigen contains one or more HLA class II peptides recognized by Tcells. One can search the sequence of the cancer associated antigenpolypeptides for HLA class H motifs as described above. In contrast toclass I peptides, class II peptides are presented by a limited number ofcell types. Thus for these experiments, dendritic cells or B cell cloneswhich express HLA class II molecules preferably are used.

TABLE 4 Sequence homologies SEQ ID NO:15 (NY-SCLC-13 5′ SEQUENCE)AL133561.1, AC007324.53, AP000552.1, AP000550.1, AC007708.13,AC009288.12, AC007325.49, AC008103.23, AC008079.22, AC008018.18,AC007731.11, AC005500, AC012398.3, AC008132.33, AC011718.2, AL117481.1,AE001958.1, AJ243721.1, X70255, X54676, AL110383.1, AL041090.1,AW261390.1, AI904151.1, AA314127, H29680, H08571, R60682, R54134,R50027, R19696, R18168, R12223, F13183, F12174, F07553, F07164, F05322,F05321, F05267, F05235, T33549, Z43231, AI828436.1, AW226624.1,AW012831.1, AW012161.1, AI874452.1, AI391139, AI225578, AI099322,AA510280, AA475860, AA276058, AA277960, AA239475, AA139948, AA106968,AA073333, AA066928, AA002337, W18896, W07975, AW148528.1, AI934011.1,AI885936.1, AI885982.1, AI824746.1, AI801523.1, AI741661.1, AI679504.1,AI589998.1, AI567632.1, AI564170.1, AI520793.1, AA677535, AA292543,F06393, AW142285.1, AW140928.1, AA520277. SEQ ID NO:16 (NY-SCLC-133′ SEQUENCE) AL133561.1, AC007324.53, AP000552.1, AP000550.1,AC007708.13, AC009288.12, AC007325.49, AC008103.23, AC008079.22,AC008018.18, AC005500, AC007731.11, AC012398.3, AC008132.33, AC011718.2,AL117481.1, AE001958.1, X70255, X54676, AF022185, U00016, AL110383.1,AL041090.1, AW261390.1, AI904151.1, AW012161.1, AI391139, AI741661.1,AW142285.1, AW140928.1, AA520277. SEQ ID NO:17 (NY-SCLC-14) X14112,D10879, Z68873.1, AJ009970.1, AF077000, M11043, AC004093, L04961,AC008124.8, AC005742, AC000395, AL023802.1, U44088, AL031258.8, U92983,Z50194, Z63758, M55701, M80829, AF192802.1, Z84494.1, AC005387,AC004490, Z93784.1, AC003976, M69157, AL031864.1, M11041, AF131866.1,AL023284.1, AF039833, U62317, NM_003980.1, AF132809.1, NM_003632.1,U38195, U38193, S44199, AB000634, NM_003459.1, NM_006245.1, D78360,AC004471, U04357, L77570, U52112, M97881, L22206, NM_004565.1,AB018269.1, AE001198, AF022844, Z82173.2, AF167560.1, AC007032.2,AB020714.1, AF037372, AC002984, U81524, U63850, Z64726, X80330,AL110210.1, AL096857.1, AL031597.7, AL021579.1, AC005932, M63138,M28265, X80327, L14589, AC011718.2, Z92546.2, AC008018.18, AP000353.1,AC004148, Y08701, AF023268, U77716, U46921, U46920, AC006549.27,Z99757.12, AC005817.6, AL035090.10, AC003063.7, AC004828.2, AC006547.9,AC000097, AF051345, Z94162.1, U34879, M84472.1, AF190826.1, M73779,AC002094, AW001248.1, AI863828.1, AI858055.1, AI813670.1, AI684429.1,AI277482, AI580934.1, AA472637, W64993, AW043820.1, AW028151.1,AI949719.1, AI887909.1, AI805058.1, AI804955.1, AI798900.1, AI741492.1,AI582191.1, AI348656, AI336325, AI299745, AI276119, AI269740, AI262960,AI200633, AI097473, AA884197, AA527274, AA480684, W68353, AI931453.1,AA726490, W98413, AW263065.1, AW211900.1, AI006238, AA255056, AA238335,H27099, AA673074, AW139762.1, AL047473.1, AW223562.1, AW066814.1,AW031777.1, AI782249.1, AI774556.1, AI586471.1, AA139570, AI923922.1,AV390350.1, AA505122, AA380178, AI853595.1, AI851994.1, AI846520.1,AI154485, AI007056, AA467529, AA274838, AA261057, AA032648, W70846,W71079, AA323008, C95416.1, AW210204.1, AA718506, AL042695.1, T25132,AI997515.1, AW205598.1, AI686223.1, AI590082.1, AI378378, AI318623,AI318236, AI201238, AI200900, AI190426, AI022738, AA916388, AA865035,AA845480, AA778028, AA744509, AA679215, AA558436, AA456062, AA418017,AA328237, AA159291, AA129371, N33970, H43255, T77577, AI890886.1,AA292501, AI379199.1, AI831459.1.Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

1. A method for diagnosing cancer, comprising contacting a biologicalsample isolated from a patient, who is suspected of having the cancer,with SOX1 (SEQ ID NO: 18) or ZIC2 (SEQ ID NO: 22) and determining thepresence or level of an antibody that binds SOX1 (SEQ ID NO: 18) or ZIC2(SEQ ID NO: 22) or antibodies that bind SOX1 (SEQ ID NO: 18) and ZIC2(SEQ ID NO: 22), wherein the presence or level of said antibody orantibodies is an indication that the subject has cancer, wherein thecancer is small cell lung cancer.
 2. The method of claim 1, wherein thesample is a body fluid, a body effusion or a tissue.
 3. The method ofclaim 2, wherein the sample is blood or serum.
 4. The method of claim 1,wherein SOX1 (SEQ ID NO:18) or ZIC2 (SEQ ID NO:22) are detectablylabeled.
 5. The method of claim 4, wherein the detectably labeled SOX1(SEQ ID NO:18) or ZIC2 (SEQ ID NO:22) is labeled with a radioactivelabel or an enzyme.
 6. The method of claim 1, wherein the nucleic acidmolecule is SOX1 (SEQ ID NO:4).
 7. The method of claim 1, wherein thenucleic acid molecule is ZIC2 (SEQ ID NO:5).
 8. The method of claim 1,wherein the sample is contacted with SOX1 (SEQ ID NO:18) and ZIC2 (SEQID NO:22).
 9. The method of claim 1, wherein the sample is contactedwith SOX1 (SEQ ID NO:18).
 10. The method of claim 1, wherein the sampleis contacted with ZIC2 (SEQ ID NO:22).